Team:Kyoto/Secretion/Notebook

From 2012.igem.org

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(→September 17)
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{{Kyoto/header}}
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==September 18==
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= Secretion Notebook =
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<div class="_kyoto-note">
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==February 7==
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'''Preculture'''<br>
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We started preculture at 12:10.<br>
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==February 8==
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'''Culture'''<br>
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We start culturing with 300[mL] of LB medium.<br>
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{|class="wikitable"
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!time||OD600
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|-
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|12:00||start
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|-
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|14:10||0.019
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|-
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|14:45||0.154
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|-
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|15:05||0.267
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|-
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|15:21||0.64
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|}
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'''Making Competent Cell'''<br>
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We made competent cells.<br><br>
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'''Transformation'''<br>
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pGEM_TAP<br>Lacp (BBa_R0011)<br>DT (BBa_B0015)<br><br>
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'''Making Culture Medium Plates'''<br>
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We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.<br><br>
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'''Transformation'''<br>
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GFP(BBa_E0040) in pSB1A2<br>DT(BBa_B0015) in pSB1AK3<br>ara(BBa_I0500) in pSB2K3<br>Lacp(BBa_R0011) in pSB1A2
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-
 
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==February 9==
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'''transformation'''<br>
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BBa-E0040(GFP)(Mr.Fujita)<br><br>
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'''Liquid culture'''<br>
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DT.leap colony transformed on February 8<br>
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colony of competent cell made on February 8<br>
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==February 10==
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'''Miniprep'''<br>
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DT2  43.9μg/ml(1.34 260/230 1.74 260/280)<br>
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lacp1 17.1μg/ml(1.54 260/280 0.83 260/230)<br>
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lacp2 18.0μg/ml(1.58 260/280 0.87 260/230)<br><br>
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'''transformation'''<br>
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B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)<br><br>
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'''Competent cell'''<br>
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We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.
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{|class="wikitable"
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!time||OD600
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|-
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|11:45||start
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|-
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|13:30||0.048
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|-
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|14:30||0.168
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|-
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|15:03||0.256
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|-
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|15:20||0.405
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|-
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|15:35||0.459
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|-
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|at last||0.576
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|}
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==February 11==
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'''Checking Transformation efficiency'''<br>
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Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg<br><br>
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==February 13==
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'''Transformation'''<br>
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Const promoter J23110,J23109,J23100<br>
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{|class="wikitable"
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!DNA||Competent cell||total
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|-
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|1μL||20||21
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|}
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No colony was there on February 14<br><br>
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'''Liquid culture'''<br>
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lacP,DT,RBS(BBa_B0034),GFP<br>
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start at 20:00<br>
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in Plus grow with Ampicilin 3mL<br>
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==February 14==
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'''Miniprep'''<br>
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concentration[μg/μL]
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{|class="wikitable"
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|lacP3||39.6
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|-
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|lacP4||40.8
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|-
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|lacP5||28.9
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|-
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|RBS1||28.2
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|-
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|RBS2||57.4
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|-
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|RBS3||13.2
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|-
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|DT3||69.7
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|-
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|DT4||64.4
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|-
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|DT5||61.5
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|-
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|GFP1||64.0
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|-
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|GFP2||50.5
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|-
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|GFP3||66.0
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|}
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'''Restriction'''<br>
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Const promoterJ23100<br>
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{|class="wikitable"
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!DNA||Spe1||Pst1||Buffer2||BSA||MilliQ||total
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|-
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|20||0.5||0.5||3||0.5||5.5||30
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|}
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at 37℃ for overnight<br>
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==February 15==
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'''Making gel'''<br>
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1% Agarose gel<br>
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{|class="wikitable"
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!Agarose||TAE
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|-
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|1.6g||160mL
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|}
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'''Electrophoresis'''<br>
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[[File:Electrophoresis021501.JPG|200px|thumb|right|No.1]]
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Gel No.1
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{|class="wikitable"
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!Restriction product||loading dye
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|-
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|5μL||1
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|}
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The marker was 1kb ladder<br>
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It seemed that this restriction product was not cut.<br><br>
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[[File:Electrophoresis021502.JPG|200px|thumb|right|No.2]]
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Gel No.2<br>
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Lane1 : 1kb ladder<br>
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Lane2 : J23100 2μL + 6*Loading dye 1μL<br>
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Lane3 : J23100(Spe1,Pst1) 5μL<br>
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Lane4 : J23100(Spe1,Pst1) 2μL<br>
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*There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.<br>
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*There must have been bands at 2100bp and 883bp on lane_3 and lane_4.<br><br>
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'''Testing whether restriction enzyme were deactivated or not'''<br>
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{|class="wikitable"
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!DNA(DT)||restriction enzyme||Buffer||BSA||MilliQ||total
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|-
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|10||0.5||3||0.5||16||30
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|}
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at 37℃ for Oveernight<br>
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Restriction enzyme means Spe1(1,2)  Pst1(1,2,3) in this time.<br><br><br><br><br><br><br><br><br><br><br><br>
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==February 16==
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'''Electrophoresis'''<br>
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[[File:Electrophoresis021601.JPG|300px|thumb|right]]
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1. 1kb ladder<br>
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2. DT2<br>
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3. DT3<br>
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4. DT2 (Spe1-1)<br>
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5. DT2 (Spe1-2)<br>
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6. DT2 (Pst1-1)<br>
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7. DT2 (Pst1-2)<br>
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8. DT2 (Pst1-3)<br>
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9. DT3 (Pst1-4)<br>
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10. 1kb ladder<br><br>
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Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.<br><br>
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'''Genomic PCR'''<br>
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{|class="wikitable"
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!10*Buffer for KOD Plus||2mM dNTPs||25mM MgSO4||10μM primer-f||10μM primer-r||158ng/μL Genomic DNA||KOD plus||MilliQ||total
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|-
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|5||5||3||1.5||1.5||1||1||32||50
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|}
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'''Electrophoresis'''<br>
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[[File:Electrophoresis021602.JPG|300px|thumb|right]]
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1. 1kb ladder<br>
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2. tatABCD (2.5kb)<br>
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3. TAMO reductase (2.7kb)<br>
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4. Negative control<br>
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We got bands of tatABCD but there were nonspecific amplification products.<br>
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We failed amplification of TAMO reductase.<br><br><br><br><br><br><br><br>
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'''Transformation'''<br>
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Constitutive promoter (BBa_J23107 , BBA_J23117)<br>
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High copy plasmid (pSB1AT3)<br>
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{|class="wikitable"
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!DNA||competent cell
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|-
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|1μL||10μL
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|}
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==February 17==
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'''PCR'''<br>
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We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.<br><br>
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'''Genomic PCR'''<br>
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{|class="wikitable"
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!Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total
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|-
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|2.5||2.5||5||0.75||0.75||0.5||0.5||16||50
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-
|}
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Predenature  94℃,  2min<br>
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Denature      98℃,  10sec<br>
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Annealing    57℃,  30sec<br>
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Extension    68℃, 2.5min<br>
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(30cycles)<br><br>
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'''Electrophoresis'''<br>
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[[File:Electrophoresis021701.JPG|300px|thumb|right]]
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1. 1kb ladder<br>
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2. TAMO reductase<br>
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3. Negative control<br><br>
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'''Restriction'''<br>
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{|class="wikitable"
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!J23100||Spe1||Pst1||Buffer2||BSA||MilliQ||total
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|-
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|10||0.5||0.5||3||0.5||15.5||30
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|}
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'''Genomic PCR'''<br>
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{|class="wikitable"
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! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total
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|-
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|TMAO reductase||2.5||2.5||3||0.75||0.75||0.5||0.5||15.5||25
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|-
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|tatABCD||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
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-
|-
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|tatABCD||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
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-
|}
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'''Electrophoresis'''<br>
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[[File:Electrophoresis021702.JPG|300px|thumb|right]]
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1. 1kb ladder <br>
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2.3.  TAMO reductase<br>
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4. tatABCD<br>
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5. tatABCD(10 times amount of genome)<br><br>
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'''Checking of restriction enzyme'''<br>
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{|class="wikitable"
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!DT||enzyme||Buffer||BSA||MilliQ||total
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|-
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|2||0.5||3||0.5||24||30
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-
|}
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at 37℃ for overnight<br>
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We checked EcoR1 and Xba1.<br><br><br><br><br><br><br><br>
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==February 18==
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'''Miniprep'''<br>
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{|class="wikitable"
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! ||μg/mL||260/280||230/260
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|-
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|JS3117-1||135||1.5||2.06
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|-
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|JS3117-2||75||1.6||1.63
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|-
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|JS3109-1||115||1.5||1.88
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|-
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|JS3109-2||75||1.65||1.71
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|-
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|pSB1AT3-1||70||1.66||1.83
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|-
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|pSB1AT3-2||100||1.52||1.54
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-
|}
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diluted to 25 times<br><br>
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'''Competent cell'''<br>
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We put 3mL of preculture product on yesterday onto 300mL of LB medium
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{|class="wikitable"
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!time||OD600
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|-
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|10:30||start
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|-
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|12:10||0.118
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|-
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|13:00||0.270
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|-
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|13:30||0.502
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|}
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'''Transformation'''<br>
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{|class="wikitable"
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!pSB1AT3-2||competent cell||MilliQ||total
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|-
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|0||20||10||30
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|-
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|2||20||8||30
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|-
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|10||20||0||30
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-
|}
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*Results(on Feb. 19)<br>
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{|class="wikitable"
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!pSB1AT3-2||number of colony
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|-
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|0||0
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|-
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|2||177
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|-
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|10||590
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-
|}
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Transformation efficiency      7.4x10^4 colonys/μg<br>
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-
 
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==February 20==
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'''Restriction'''<br>
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sample 1<br>
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{|class="wikitable"
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!DT plasmid||EcoR1||Xba1||Buffer||BSA||MilliQ||total
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-
|-
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|7.5||0.5||0.5||3||0.5||18||30
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-
|}
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sample2<br>
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{|class="wikitable"
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!GFP plasmid||EcoR1||Spe1||Buffer||BSA||MilliQ||total
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-
|-
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|10||0.5||0.5||3||0.5||15.5||30
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-
|}
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'''Electrophoresis'''<br>
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*sample1<br>
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[[File:Electrophoresis022001_.JPG|200px|thumb|right]]
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-
a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>
+
-
b. sample1 5μL + 6×Loading Dye 1μL<br>
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-
lane_1 1kb ladder<br>
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-
lane_2 a<br>
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lane_3 b<br>
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-
lane_4 a<br>
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lane_5 b<br>
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lane_6 a<br>
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lane_7 b<br>
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lane_8 1kb ladder<br>
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*sample2<br>
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[[File:Electrophoresis022302.JPG|200px|thumb|right]]
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-
c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>
+
-
d. sample2 5μL + 6×Loading Dye 1μL<br>
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-
lane_1 1kb ladder<br>
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-
lane_2 c<br>
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-
lane_3 d<br>
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-
lane_4 1kb ladder<br><br>
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-
'''PCR'''<br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total
+
-
|-
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|tatABCD1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|-
+
-
|tatABCD2||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|-
+
-
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
+
-
|-
+
-
|TMAO reductase2||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
+
-
|}
+
-
Predenature  94℃    2min<br>
+
-
Denature      98℃    10sec<br>
+
-
Annealing    59℃    30sec<br>
+
-
Extension    68℃    2.5min<br>
+
-
→30cycles<br><br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||PCR products||genomic DNA||KOD plus||MilliQ||total
+
-
|-
+
-
|tatABCD1||2.5||2.5||1.5||0.75||0.75||0||0.5||0.5||16||25
+
-
|-
+
-
|tatABCD2||2.5||2.5||1.5||0.75||0.75||1||0||0.5||15.5||25
+
-
|-
+
-
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25
+
-
|-
+
-
|TMAO reductase2||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25
+
-
|}
+
-
Predenature  94℃    2min<br>
+
-
Denature    98℃    10sec<br>
+
-
Annealing    59℃    30sec<br>
+
-
Extension    68℃  2.5min<br>
+
-
→35cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022003.JPG|200px|thumb|right]]
+
-
lane_1.1kb ladder<br>
+
-
lane_2.tatABCD<br>
+
-
lane_3.tatABCD<br>
+
-
lane_4.TAMO1<br>
+
-
lane_5.TAMO2<br>
+
-
lane_6.1kb ladder<br><br><br><br><br>
+
-
[[File:Electrophoresis022004.JPG|200px|thumb|right]]
+
-
lane_1.1kb ladder<br>
+
-
lane_2.tatABCD1<br>
+
-
lane_3.tatABCD2<br>
+
-
lane_4.TAMO<br>
+
-
lane_5.TAMO<br>
+
-
lane_6.1kb ladder<br><br><br><br><br><br>
+
-
 
+
-
==February 21==
+
-
'''PCR (Advantage HF protocol)'''<br>
+
-
{|class="wikitable"
+
-
! ||buffer||dNTPs||primer-f||primer-r||gDNA||PCR products||DW||polymerase||total
+
-
|-
+
-
|tatABCD||2.5||2.5||0.75||0.75||1||0||17||0.5||25
+
-
|-
+
-
|TMAO||2.5||2.5||0.75||0.75||0||1||17||0.5||25
+
-
|}
+
-
Predenature  94℃    1min<br>
+
-
Denature    94℃    30sec<br>
+
-
Annealing    58℃    30sec<br>
+
-
Extension    68℃    3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis0221.JPG|250px|thumb|right]]
+
-
1. 1kb ladder  2μL<br>
+
-
2. tatABCD  5μL + 6×Loading Buffer  1μL<br>
+
-
3. TAMO  5μL + 6×Loading Buffer  1μL<br>
+
-
4. 1kb ladder  2μL<br><br>
+
-
'''Liquid culture'''<br>
+
-
pSB3C5-1,2<br>
+
-
pSB4K5-1,2
+
-
 
+
-
==February 22==
+
-
'''Gel extraction'''<br>
+
-
lane 1 of the gel 45.0μg/mL<br><br>
+
-
'''PCR purification'''<br>
+
-
product  38.2μg/mL <br><br>
+
-
'''PCR'''<br>
+
-
TMAO reductase<br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of gel extract||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total
+
-
|-
+
-
|1||5||5||4||1.5||1.5||0.5||0||1||32.5||50
+
-
|-
+
-
|2||5||5||4||1.5||1.5||1||0||1||32.5||50
+
-
|-
+
-
|3||5||5||4||1.5||1.5||0||0.5||1||32.5||50
+
-
|-
+
-
|4||5||5||4||1.5||1.5||0||1||1||32.5||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
59℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''
+
-
[[File:Electrophoresis022201.JPG|250px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TAMO1<br>
+
-
3. TAMO2<br>
+
-
4. TAMO3<br>
+
-
5. TAMO4<br>
+
-
6. constructive promoter 1-18C<br>
+
-
7. constructive promoter Spe1<br>
+
-
8. constructive promoter Pst1<br>
+
-
9. 1kb ladder<br>
+
-
'''PCR'''<br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total
+
-
|-
+
-
|1||5||5||3||1.5||1.5||0.5||1||32.5||50
+
-
|-
+
-
|2||5||5||3||1.5||1.5||1||1||32||50
+
-
|-
+
-
|3||5||5||3||1.5||1.5||2||1||31||50
+
-
|-
+
-
|4||5||5||3||1.5||1.5||3||1||30||50
+
-
|-
+
-
|5||5||5||3||1.5||1.5||10||1||29||50
+
-
|-
+
-
|6||5||5||3||1.5||1.5||0||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
59℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022202.JPG|300px|thumb|right]]<br><br><br><br><br><br><br><br><br><br><br><br>
+
-
'''Checking Dpn1'''<br>
+
-
{|class="wikitable"
+
-
!Buffer||lacp(28.7ng/μL)||Dpn1||MilliQ||total
+
-
|-
+
-
|2||10||0.5||7.5||20
+
-
|-
+
-
|2||10||0||5||17
+
-
|}
+
-
 
+
-
==February 23==
+
-
'''Colony PCR'''<br>
+
-
tatABCD(2 samples)<br>
+
-
{|class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||KOD plus||MilliQ||total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
 
+
-
Predenature 94℃  1min<br>
+
-
Denature    98℃ 10sec<br>
+
-
Annealing  59℃ 30sec<br>
+
-
Extension  68℃  3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022301.JPG|300px|thumb|right]]
+
-
1. 1kb ladder 2μL<br>
+
-
2. tatABCD 1  5μL + 6×Loading Buffer  1μL<br>
+
-
3. tatABCD 2  5μL + 6×Loading Buffer  1μL<br>
+
-
4. 1kb ladder 2μL<br><br>
+
-
'''Miniprep'''<br>
+
-
pSB4K5 and pSB3C5<br>
+
-
deluted it to 25 times and then measured it<br>
+
-
pSB4K5 1 : 60.0 μg/ml      1.67(260/280)      1.98(260/230) <br>
+
-
pSB4K5 2 : 55.0 μg/ml      1.49(260/280)      1.62(260/230) <br>
+
-
pSB3C5-3 : 3.6 μg/ml        1.57(260/280)      3.00(260/230) <br>   
+
-
pSB3C5-4 : 1.3 μg/ml        1.44(260/280)      1.04(260/230) <br><br>
+
-
'''Liquid culture'''<br>
+
-
pSB3C5-3,4<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022302.JPG|300px|thumb|right]]
+
-
1. 1kb ladder 2μL<br>
+
-
2. pSB3C5-3 5μL, 6×loading dye 1μL<br>
+
-
3. pSB3C5-4 5μL, 6×loading dye 1μL<br>
+
-
4. 1kb ladder 2μL<br><br><br><br><br><br><br><br>
+
-
 
+
-
==February 27==
+
-
'''Test of Dpn1'''<br>
+
-
{| class="wikitable"
+
-
!Buffer2||GFP2||BSA||MilliQ||Dpn1
+
-
|-
+
-
|3||3||0.3||23||1
+
-
|}
+
-
'''Colony PCR'''<br>
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||MilliQ||KOD Plus||total
+
-
|-
+
-
|Colony PCR(2 samples)||5||5||3||1.5||1.5||33||1||50
+
-
|-
+
-
|Negative control||5||5||3||1.5||1.5||34||0||50
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis0227.JPG|200px|thumb|right]]
+
-
{| class="wikitable"
+
-
! ||sample||Loading Dye||MilliQ
+
-
|-
+
-
|1.1kb ladder||2||0||0
+
-
|-
+
-
|2.product of PCR1||5||1||0
+
-
|-
+
-
|3.product of PCR2||5||1||0
+
-
|-
+
-
|4.product of PCR(Negative control)||5||1||0
+
-
|-
+
-
|5.product of PCR(2/23)||5||1||0
+
-
|-
+
-
|6.GFP2(DPN1)||10||2||0
+
-
|-
+
-
|7.GFP2||3||2||7
+
-
|-
+
-
|8.1kb ladder||2||0||0
+
-
|}
+
-
'''Results of liquid culture'''<br>
+
-
We measure this after dilute it to 10 times.<br>
+
-
{|class="wikitable"
+
-
!pSB3C5-5||pSB3C5-6||pSB3C5-5(1% glucose)||pSB3C5-6(1% glucose)
+
-
|-
+
-
|8.5[µg/ml]||-1.8||-17.9||-18.2
+
-
|}
+
-
'''PCR'''<br>
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f(prefix)||Primer-r(suffix)||PCR purification product(1ng/µL)||MilliQ||KOD Plus||total
+
-
|-
+
-
|1||5||5||3||1.5||1.5||0.2||32.8||1||50
+
-
|-
+
-
|2||5||5||3||1.5||1.5||0.5||32.5||1||50
+
-
|}
+
-
*PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL<br>
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br><br>
+
-
 
+
-
==February 28==
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022801.JPG|250px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]<br>
+
-
3. PCR2<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!Buffer2||plasmid(?)||enzyme||MilliQ||total
+
-
|-
+
-
|2||2||0.2||15.8||20
+
-
|}
+
-
incubate 1 hour at 37℃<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022802.JPG|250px|thumb|right]]
+
-
[[File:Electrophoresis022803.JPG|250px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. Control (without enzymes)<br>
+
-
3. EcoR1<br>
+
-
4. Xba1 (crystallized)<br>
+
-
5. Xba1 (with seal)<br>
+
-
6. Spe1<br>
+
-
7. Pst1<br>
+
-
8. 1kb ladder<br><br>
+
-
'''PCR and Electrophoresis'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq Dye Mix||primer-f||primer-r||template||MilliQ||total
+
-
|-
+
-
|25||1.0||1.0||0.5||22.5||50
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 94℃,30sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!BufferH||tatABCD||EcoR1||Spe1||MilliQ||total
+
-
|-
+
-
|2||5||0.2||0.2||12.6||20
+
-
|}
+
-
'''PCR purification'''<br>
+
-
We eluted the product for 30µL MilliQ<br><br>
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Insert(tatABCD)||Vector(pSB1C3)||Ligation High||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
4℃, overnight
+
-
 
+
-
==February 29==
+
-
'''Transformation'''<br>
+
-
{|class="wikitable"
+
-
!tatABCD||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
'''Checking Restriction enzyme'''<br>
+
-
{|class="wikitable"
+
-
!plasmid seems to be 1-18C promoter||Enzyme||Buffer||MilliQ||total
+
-
|-
+
-
|2||0.2||2||15.8||20
+
-
|}
+
-
'''Checking tatABCD'''<br>
+
-
{| class="wikitable"
+
-
!tatABCD||Hind3||Buffer||MilliQ||total
+
-
|-
+
-
|5||0.2||2||12.8||20
+
-
|}
+
-
'''PCR'''<br>
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||ColE1(6.5ng/µL) / TMAO||MilliQ||KOD Plus Neo||total
+
-
|-
+
-
|Kil||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|-
+
-
|TMAO||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 60℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→30cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
*evernoteに写真なし！至急求む
+
-
1. 1kb ladder<br>
+
-
2. Kil (649bp)<br>
+
-
3. TMAO (2720bp)<br>
+
-
4. TMAO (Quick Taq)<br>
+
-
5. tatABCD (Quick Taq)<br>
+
-
6. tatABCD (Hind3)<br>
+
-
7. 1kb ladder<br>
+
-
 
+
-
==March 1==
+
-
'''PCR'''<br>
+
-
*TMAO<br>
+
-
Template is gDNA and product of colony PCR gel extraction<br>
+
-
{| class="wikitable"
+
-
! ||Buffer||gNTPs||MgSO4||Primer-f||Primer-r||KOD Plus Neo||Template gDNA||product of gel extraction||DW||total
+
-
|-
+
-
|1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||0||16||25
+
-
|-
+
-
|2||2.5||2.5||1.5||0.75||0.75||0.5||0||2||14.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→25cycles<br><br>
+
-
*Kil
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||colE1(6.5ng/µL)||KOD Plus Neo||MilliQ||total
+
-
|-
+
-
|2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→20cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis030101.JPG|250px|thumb|right]]
+
-
[[File:Electrophoresis030102.JPG|250px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TMAO1 (gDNA)<br>
+
-
3. TMAO2 (product of gel extraction)<br>
+
-
4. Kil<br><br>
+
-
'''PCR'''<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||Product of Purification||KOD Plus Neo||MilliQ||total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
20cycles<br>
+
-
→Purification 230ng/µL<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!Kil||EcoR1||Spe1||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||2||12.6||20
+
-
|}
+
-
incubate at 37℃, for 1.5 hours<br><br>
+
-
'''PCR Purification'''<br><br>
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 4℃, for overnight
+
-
 
+
-
==March 2==
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!tatABCD||pSB1C3||Ligation||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
'''Transformation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||Kil(3/1,Ligation)||tatABCD||competet cell||total
+
-
|-
+
-
|1||0||0||10||11
+
-
|-
+
-
|0||1||0||10||11
+
-
|-
+
-
|0||0||1||10||11
+
-
|}
+
-
'''PCR'''
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-r||primer-f||template||MilliQ||total
+
-
|-
+
-
|25||1||1||0.5||22.5||50
+
-
|}
+
-
'''Electrophoresis'''
+
-
[[File:Electrophoresis030227.JPG|250px|thumb|right]]
+
-
<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!pSB3C5-5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃, for 2 hour<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis030225.JPG|250px|thumb|right]]
+
-
1. 1kb ladder 2µL<br>
+
-
2. pSB3C5 5µL + 6×Loading Buffer 1µL<br>
+
-
・product of gel extraction(about 2700bp)<br>
+
-
－30.9µg/mL<br><br>
+
-
'''Restriction'''<br>
+
-
GFP1,2,3
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Pst1||Buffer||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃, for 2.5 hours<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Constitutive Promoter||Spe1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||0.2||0.2||3||0.3||11.3||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
*J23117-1:135ng/µL, J23107-1:115ng/µL<br>
+
-
*DT3→PCR Purification<br>
+
-
*Promoter→Gel Extraction<br><br>
+
-
'''Checking TMAO'''<br>
+
-
{| class="wikitable"
+
-
!something seems to be TMAO||Buffer2||EcoR1||MilliQ||total
+
-
|-
+
-
|10||2||0.5||7.5||20
+
-
|}
+
-
at 37℃, for 1 hour<br><br>
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis030226.JPG|250px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. GFP1 that had been cut by restriction enzyme<br>
+
-
3. GFP2 that had been cut by restriction enzyme<br>
+
-
4. GFP3 that had been cut by restriction enzyme<br>
+
-
5. GFP1<br>
+
-
6. GFP2<br>
+
-
7. GFP3<br>
+
-
8. TMAO (control)<br>
+
-
9. TMAO (EcoR1)<br>
+
-
10. DT (control)<br>
+
-
11. DT (EcoR1, Xba1)<br>
+
-
12. 1kb ladder<br><br>
+
-
'''Checking tatABCD'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-f||primer-r||MilliQ||total
+
-
|-
+
-
|25||1||1|||23||50
+
-
|}
+
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis030228.JPG|250px|thumb|right]]
+
-
<br><br><br><br><br><br><br><br><br><br><br><br>
+
-
 
+
-
==March 3==
+
-
====PCR====
+
-
{| class="wikitable" style="text-align: right"
+
-
! ||template||buffer||dNTPs||MgSO4||VF||VR||KOD plus||MilliQ||total
+
-
|-
+
-
|1||1||5||5||3||1.5||1.5||1||32||50
+
-
|-
+
-
|2||2||5||5||3||1.5||1.5||1||31||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
50℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→30cycles<br><br>
+
-
====Miniprep====
+
-
 
+
-
==March 4==
+
-
====Sequence of tatABCD====
+
-
{| class="wikitable" style="text-align: right;"
+
-
!Quick Taq||primer-f||promer-r sequence||template||MilliQ||total
+
-
|-
+
-
|25||1||1||1||23||50
+
-
|}
+
-
{| class="wikitable" style="text-align: right;"
+
-
!Quick Taq||primer-f sequence||primer-r||template||MilliQ||total
+
-
|-
+
-
|25||1||1||1||23||50
+
-
|}
+
-
====Colony PCR of TMAO====
+
-
{| class="wikitable" style="text-align: right;"
+
-
!buffer||dNTPs||NgSO4||primer-f||primerr-r||KOD plus||MilliQ||total
+
-
|-
+
-
|5||5||4||1.5||1.5||1||32||50
+
-
|}
+
-
→ethanol precipitation<br>
+
-
 
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 2.5min<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
1. 1kb ladder<br>
+
-
2. tatABCD1<br>
+
-
3. tatABCD2<br>
+
-
4. TMAO<br>
+
-
5. 1kb ladder<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!TMAO||EcoR1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||3||0.3||16.5||30
+
-
|}
+
-
{|class="wikitable"
+
-
!TMAO||Xba1||Pst1||BudderM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
at 37℃ for 1 hour<br><br>
+
-
====Electrophoresis====
+
-
<br>
+
-
 
+
-
====Transformation====
+
-
{|class="wikitable"
+
-
!pSB1C3||competent cell(made at 2/8)||total
+
-
|-
+
-
|5||100||105
+
-
|}
+
-
 
+
-
==March 5==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3(Xba1, Spe1)||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|-
+
-
!pSB1C3(Xba1, Spe1)||EcoR1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|}
+
-
at 37℃ for 1 hour<br>
+
-
→Then we did ethanol precipitation<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil(EcoR1, Spe1)||pSB1C3(EcoR1)||Ligation High||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Kil||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
We used commercially available competent cells in this time.<br><br>
+
-
====PCR====
+
-
TMAO<br>
+
-
{| class="wikitable"
+
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||Template||KOD plus||MilliQ||total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||1||32||50
+
-
|}
+
-
====Electrophoresis====
+
-
(31)<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
! ||Lacp||pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|1||20||0||0.5||0.5||3||0.5||5.5||30
+
-
|-
+
-
|2||0||20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃  for 1 hour<br>
+
-
1→Ethanol precipitation　45.4µg/mL<br>
+
-
2→Gel extraction　38.7µg/mL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!LacP||pSB3C5||Ligation High||total
+
-
|-
+
-
|10||2||6||18
+
-
|}
+
-
at 4℃ for overnight<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Lacp+pSB3C5||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
on ice for 30 mins.<br>
+
-
heat shock at 42℃ for 60secs<br>
+
-
on ice for 2 mins.<br>
+
-
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!GFP Plasmid||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 2 hours
+
-
====Electrophoresis====
+
-
1. Ladder 2µL<br>
+
-
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL<br>
+
-
3. Ladder 2µL<br>
+
-
====PCR====
+
-
torA signal and pspA
+
-
pspAはコロニーPCR<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||Primer-f||Primer-r||template(TMAO)||KOD plus||MilliQ||total
+
-
|-
+
-
|2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
====electrophoresis====
+
-
1. 100bp Ladder<br>
+
-
2. torA signal (272bp)<br>
+
-
3. pspA (969bp)<br>
+
-
4. 100bp Ladder<br>
+
-
 
+
-
==March 6==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|12||0.5||0.5||3||0.5||13.5||30
+
-
|-
+
-
|}
+
-
We did incubate at 37℃ for 1.5hours.<br>
+
-
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.<br><br>
+
-
====PCR====
+
-
{| class="wikitable"
+
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||template||KOD plus neo||MilliQ||total
+
-
|-
+
-
|5||5||3||1.5||1.5||0.5||1||32.5||50
+
-
|}
+
-
94℃　2min<br>
+
-
98℃　10sec<br>
+
-
60℃　30sec<br>
+
-
68℃　(torA 10sec / pspA 30sec)    25 cycles<br>
+
-
We used product of PCR on 3/5 of torA and pspA as template.<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!Kil||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.3||0.3||3||0.3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 2 hours<br>
+
-
→ purification 37.7ng/μL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
*Kil : 350fmol<br>
+
-
*pSB1C3 : 29fmol<br>
+
-
at 16℃ for overnight<br>
+
-
 
+
-
==March 7==
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
+
-
1. 1kb ladder 2µL<br>
+
-
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL<br>
+
-
3. GFP 30µL + Loading Dye 6µL<br>
+
-
4. 1kb ladder 2µL<br>
+
-
*The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!VectorDNA||GFP||Ligation High Ver.2||total
+
-
|-
+
-
|5||15||10||30
+
-
|}
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.3||0.3||3||0.3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.3||0.3||3||0.3||21.1||30
+
-
|}
+
-
at 37℃ for 1.5 hours<br><br>
+
-
====Purification====
+
-
torA→31.8ng/µL<br>
+
-
pspA→49.3ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|3||3||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 4℃, for overnight<br>
+
-
*torA→31.8ng/µL×3µL=95.4ng=0.529pmol<br>
+
-
*pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol<br>
+
-
*pspA→49.3ng/µL×4µL=197.2ng=0.308pmol<br>
+
-
*pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol<br>
+
-
 
+
-
==March 8==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.2||0.2||3||0.2||6.4||30
+
-
|-
+
-
|}
+
-
at 37℃ for 1 hour.<br>
+
-
→Purification : 36.6ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB4K5||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 4℃ for overnight<br>
+
-
*Kil→37.7ng/µL×10µL=377ng=879fmol<br>
+
-
*pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
+
-
====Liquid culture====
+
-
Lacp + pSB3C5 -1, 2<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!torA||pspA||competent cell||total
+
-
|-
+
-
|1||0||10||11
+
-
|-
+
-
|0||1||10||11
+
-
|}
+
-
We use commercially available competent cells in this time.<br>
+
-
 
+
-
==March 9==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
at 37℃ for 1hour<br>
+
-
→purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
+
-
====Miniprep====
+
-
lacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
+
-
lacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
+
-
====Colony PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃ 2min<br>
+
-
94℃ 30sec<br>
+
-
55℃ 30sec<br>
+
-
68℃ 6sec<br>
+
-
25cycles<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!tatABCD||constP J23107||Ligation High Ver.2||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
tatABCD : 227fmol<br>
+
-
constP J23107 : 21fmol<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
! ||pspA||torA||Kil||competent cell
+
-
|-
+
-
|1||1||0||0||10
+
-
|-
+
-
|2||0||1||0||10
+
-
|-
+
-
|3||0||0||1||10
+
-
|}
+
-
====Miniprep====
+
-
4mL of plusgrow which had been cultured for overnight.<br>
+
-
pSB4K5 : 80.5μg/mL<br>
+
-
 
+
-
==March 10==
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
[[File:Electrophoresis0310.JPG|400px|thumb|right]]
+
-
Then we did electrophoresis to confirm.<br>
+
-
1.1kb ladder<br>
+
-
2.kil (649bp)<br>
+
-
3,4,5, pspA (969bp)<br>
+
-
6,1kb ladder<br><br>
+
-
1, 100bp ladder<br>
+
-
2,3,4, torA signal<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
-
for 2.5 hours at 37℃<br>
+
-
→purification  29.0ng/μL<br>
+
-
{| class="wikitable"
+
-
!torA||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
-
for 2.5 hours at 37℃<br>
+
-
→purification  91.8ng/μL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||Lacp-pSB3C5||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
torA : 929fmol<br>
+
-
Lacp-pSB3C5 : 284fmol<br>
+
-
for overnight at 4℃
+
-
 
+
-
==March 11==
+
-
====Miniprep====
+
-
We used 3μL of plus grow that we had cultured for overnight.<br>
+
-
torA : 62.8ng/μL<br><br>
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis031101.JPG|400px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2〜11. pspA (969bp)<br>
+
-
12. 1kb ladder<br>
+
-
<br>The results were shown as photograph in the right.<br>
+
-
<br>It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.<br><br><br><br><br><br>
+
-
[[File:Electrophoresis031102.JPG|400px|thumb|right]]<br>
+
-
1.1kb ladder<br>
+
-
2. pspA (PCR product)<br>
+
-
3, pspA (Eco, Spe)<br>
+
-
4〜6, pspA (colony PCR)<br>
+
-
7,1kb ladder<br>
+
-
<br>The results were shown as photograph in the right.<br>
+
-
 
+
-
==March 12==
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!DT(1ng/μL)||DT(0.1ng/μL)||Kil||lacP-torA||MilliQ||competent cell||total
+
-
|-
+
-
|1||0||0||0||0||20||21
+
-
|-
+
-
|0||1||0||0||0||20||21
+
-
|-
+
-
|0||0||5||0||0||50||51
+
-
|-
+
-
|0||0||0||5||0||50||51
+
-
|-
+
-
|0||0||0||0||1||20||21
+
-
|}
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.3||15.7||30
+
-
|}
+
-
at 37℃ for 4 hours<br>
+
-
→ We did purification and got 48.3ng/μL pspA.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||total
+
-
|-
+
-
|1|4||2||0||3||9
+
-
|-
+
-
|2|2||2||0||2||6
+
-
|-
+
-
|3|0||2||2||2||6
+
-
|}
+
-
 
+
-
==March 13==
+
-
====Miniprep====
+
-
pSB1C3  74.2µg/mL  1.65 (260/280)  1.31 (260/230)
+
-
==March 14==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.2||0.2||4||0.4||15.2||40
+
-
|}
+
-
We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|-
+
-
!MilliQ||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 16℃, for 1 hour<br>
+
-
*torA : 0.707pmol<br>
+
-
*pSB1C3 : 0.068pmol<br><br>
+
-
====Liquid culture====
+
-
We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.<br>
+
-
→Only 4 which did not be cultured succeeded.
+
-
 
+
-
==March 15==
+
-
====Liquid culture====
+
-
We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
+
-
→6,8,9,10 were succeeded.<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
for 2 hours at 37℃.<br><br>
+
-
====Miniprep====
+
-
pspA (pSB1C3)  40.5ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||total
+
-
|-
+
-
|5||1.5||3||9.5
+
-
|}
+
-
*pspA : 385fmol<br>
+
-
*DT : 36fmol<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||DT (0.1ng/µL)||DT (0.01ng/µL)||pspA-DT||competent cells on 3/15||total
+
-
|-
+
-
|2||0||0||0||20||22
+
-
|-
+
-
|0||2||0||0||20||22
+
-
|-
+
-
|0||0||2||0||20||22
+
-
|-
+
-
|0||0||0||2||20||22
+
-
|}
+
-
====Screening PCR====
+
-
Kil, pspA and torA<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-r||Primer-f||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
 
+
-
==March 16==
+
-
====Miniprep====
+
-
torA (pSB1C3)  68.8ng/µL<br>
+
-
Kil (pSB4K5)  92.7ng/µL<br>
+
-
torA was red for some reason. We do not know why.<br><br>
+
-
====Colony PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-r||Primer-f||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 6sec<br>
+
-
→25cycles<br><br>
+
-
====Electrophoresis====
+
-
[[File:Electrophoresis0316.JPG|400px|thumb|right]]
+
-
The results were shown as photograph in the right.<br><br>
+
-
====Checking Transformation Efficiency====
+
-
competent cells that were made on March 15.<br>
+
-
DNA : 0.02ng → 668 colonies  Transformation Efficiency : 3.3×10^7<br>
+
-
DNA : 0.2ng → 1739 colonies  Transformation Efficiency : 8.7×10^6<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||BufferH||MilliQ||total
+
-
|-
+
-
|20||0.2||0.2||0.3||3||0||6.3||30
+
-
|-
+
-
|5||0.2||0||0.2||0||2||12.6||20
+
-
|}
+
-
at 37℃, for 2 hours.<br>
+
-
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.<br>
+
-
{| class="wikitable"
+
-
!Kil(pSB4K5)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
for overnight at 37℃.<br>
+
-
We did this to confirm.<br>
+
-
{| class="wikitable"
+
-
!pspA (pSB1C3)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
for overnight at 37℃.<br>
+
-
We did this to confirm.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!MilliQ||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 16℃, for overnight<br>
+
-
*torA→767fmol<br>
+
-
*pSB1C3→68fmol<br>
+
-
 
+
-
==March 17==
+
-
====Miniprep====
+
-
J23107-tatABCD  72.7ng/µL<br>
+
-
pspA-DT  50.5ng/µL<br><br>
+
-
====Checking the Insert====
+
-
{| class="wikitable"
+
-
!J21037-tatABCD||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Success.
+
-
{| class="wikitable"
+
-
!pspA-DT||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Failed.
+
-
 
+
-
 
+
-
 
+
-
==March 19==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did Gel extraction and got 17.2ng/µL of DT.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||DT||Ligation High Ver.2||total
+
-
|-
+
-
|10||2||6||18
+
-
|}
+
-
We did this for an hour at 16℃.<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||0.5||3||15.5||30
+
-
|}
+
-
We did this for 4 hours at 37℃<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||total
+
-
|-
+
-
|5||5||5||15
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||1||3||8
+
-
|}
+
-
We did these for an hour at 16℃.<br>
+
-
*pspA (5µL)→377fmol<br>
+
-
*DT→39fmol<br>
+
-
*pspA (4µL)→339fmol<br>
+
-
*pSB1C3→34fmol<br><br>
+
-
====Transformation====
+
-
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
+
-
 
+
-
==March 20==
+
-
====Screaning PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-R||Primer-F||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→○<br>
+
-
*GFP-DT→○<br>
+
-
*torA→×<br>
+
-
*Kil-DT 6 of 8 sumples→○<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VR||VF||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→×<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.3||0.3||0.3||3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did this for overnight at 37℃. And we did purification.<br>
+
-
torA  34.2ng/µL<br>
+
-
DT  28.0ng/µL<br><br>
+
-
 
+
-
==March 21==
+
-
====Miniprep====
+
-
GFP-DT-1 : 77.7ng/µL<br>
+
-
GFP-DT-2 : 67.6ng/µL<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3<br><br>
+
-
{| class="wikitable"
+
-
!GFP-DT||EcoR1||Pst1||Xba1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||0.2||2||12.6||20
+
-
|-
+
-
|10||0.2||0||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSB1C3||pspA||DT||GFP-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||4||1||0||0||0||3||8
+
-
|-
+
-
|2||0||1||7||0||0||4||12
+
-
|-
+
-
|3||0||0||5||3||0||4||12
+
-
|-
+
-
|4||3||0||0||0||5||4||12
+
-
|}
+
-
We did this for an hour at16℃.
+
-
 
+
-
==March 22==
+
-
====PCR====
+
-
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||Primer-F||Primer-R||Template||MilliQ||KOD plus neo||total
+
-
|-
+
-
|5||5||3||1.5||1.5||0.5||32.5||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 10sec<br>
+
-
→30cycles<br>
+
-
→Purification  110.7ng/µL<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSSB1C3||pspA||DT||GFP-DT||Ligation high||total
+
-
|-
+
-
|1||4||3||0||0||0||4||11
+
-
|-
+
-
|2||3||0||0||0||3||3||9
+
-
|-
+
-
|3||0||0||5||5||0||5||15
+
-
|}
+
-
*torA (4µL)→512fmol<br>
+
-
*pSB1C3→54fmol<br>
+
-
*torA (3µL)→384fmol<br>
+
-
*GFP-DT→36fmol<br>
+
-
*pspA→377fmol<br>
+
-
*DT→65fmol<br><br>
+
-
 
+
-
==March 23==
+
-
====Screening PCR====
+
-
torA (pSB1C3), torA-GFP-DT and pspA-DT<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.3||0.3||3||0.3||21.1||30
+
-
|}
+
-
And then we did ethanol precipitation<br><br>
+
-
====Ethanol precipitation====
+
-
pspA 11.5ng/µL.<br><br>
+
-
====Miniprep====
+
-
Lacp+pSB3C5-8  77.9µg/mL<br>
+
-
Lacp+pSB3C5-10  69.0µg/mL<br>
+
-
Kil+DT-4  58.0µg/mL<br>
+
-
Kil+DT-7  15.2µg/mL<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!Lacp+pSB3C5-8||Spe1||Pst1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Kil+DT-4||Xba1||Pst1||Buffer2||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1.5 hours<br>
+
-
And then we did Gel extraction.<br><br>
+
-
====Gel Extraction====
+
-
Lacp+pSB3C5-8  40.4µg/mL<br>
+
-
Kil+DT-4  26.8µg/mL<br>
+
-
 
+
-
==March 26==
+
-
====Miniprep====
+
-
torA(pSB1C3) 18.5[ng/µL]<br>
+
-
torA-GFP-DT 20.0[ng/µL]<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA(pSB1C3)||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||2||0.2||12.4||20
+
-
|-
+
-
|5||0.2||0||2||0.2||12.6||20
+
-
|}
+
-
{| class="wikitable"
+
-
!torA-GFP-DT||EcoR1||Xba1||Pst1||BufferH||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.2||0||0.2||2||0||0.2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||2||0||0.2||12.6||20
+
-
|-
+
-
|20||0||0.2||0.2||0||3||0.3||6.3||30
+
-
|}
+
-
We did Gel extraction and then got ??? 28.7[ng/µL]<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Lacp (pSB3C5)||torA-GFP-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
*Lacp : 22fmol<br>
+
-
*torA-GFP-DT : 197fmol<br>
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
*pspA : 180fmol<br>
+
-
*pSB1C3 : 18fmol<br>
+
-
*DT : 16fmol<br>
+
-
{| class="wikitable"
+
-
!LacP(pSB3C5)||Kil-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
for 2 hours at 16℃<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Lacp-Kil-DT||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
 
+
-
==March 27==
+
-
====Miniprep====
+
-
We retryed miniprep of torA(pSB1C3).<br>
+
-
We got torA and its concentration was 39.3[ng/µL].<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
+
-
|-
+
-
|BBa_K117004 ||14J(2011 plate2)||5||20||?||?||?
+
-
|}
+
-
We added 100[µL] of culture medium before we started culturing the E.coli.<br><br>
+
-
====Screening PCR====
+
-
{| class="wikitable"
+
-
!Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.<br>
+
-
pspA-DT was failed.<br>
+
-
E.coli that had pspA(pSB1C3) did not make any colony.<br>
+
-
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.<br><br>
+
-
====Liquid culture====
+
-
Lacp-torA-GFP-DT<br>
+
-
</div>
+
-
==July 23==
+
-
====Transformation====
+
-
{|class="wikitabloe"
+
-
!DT(64.4ng/μl)||Competent cell||Plating in SOC medium||Total
+
-
|-
+
-
|1||20||100||121
+
-
|}
+
-
==July 24==
+
-
====Plating in SOC medium====
+
-
==July 25==
+
-
====Transformation====
+
-
*BBa_J23113 from iGEM Kit
+
-
 
+
-
*LacP from iGEM Kit
+
-
==July 25==
+
-
==July 26==
+
-
==July 27==
+
-
====Restriction Enzyme Processing====
+
-
{|class="wikitable"
+
-
!Kil +DT (44.0 ng/μl)||10xBuffer2||BSA||Xbal||MilliQ||Total
+
-
|-
+
-
|13.6||3.0||0.5||0.5||11.9||30.0
+
-
|}
+
-
{|class="wikitable"
+
-
!LacP(28.7ng/μl)||10xBuffer2 ||BSA||Spe1||Pst1||Total
+
-
|-
+
-
|20.9||3.0||0.5||0.5||4.6||30.0
+
-
|} 
+
-
==July 30==
+
-
 
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!kil+DT(XbaI,PstI)||LacP(SpeI,PstI)||Ligation High ||Total
+
-
|-
+
-
|30||6||36||72
+
-
|}
+
-
==July 31==
+
-
====Restriction Enzyme Processing ====
+
-
{| class="wikitable"
+
-
!torAGFP+DT ||10×M Buffer||BSA ||MilliQ||XbaI||PstI||Total
+
-
|-
+
-
|2.5||3.0||0.5||23.4||0.3||0.3||30.0
+
-
|}
+
-
====Liquid Culture====
+
-
J23113(backbone J61002)
+
-
==August 1==
+
-
====Restriction Enzyme Processing====
+
-
{|class="wikitable"
+
-
!lacP+kil+DT ||BSA||10×H Buffer||EcoRI||PstI||MilliQ||Total
+
-
|-
+
-
|5.0||0.5||3.0||0.5||0.5||20.5||30.0
+
-
|}37℃ 2h
+
-
{|class="wikitable"
+
-
!lacP middle copy ||BSA||10×H Buffer||EcoRI||PstI||MilliQ||Total
+
-
|-
+
-
|10.0||0.5||3.0||0.5||0.5||15.5||30.0
+
-
|}37℃ 2h
+
-
 
+
-
====MIniprep====
+
-
J23113(backbone J61002)
+
-
====Liquid culture====
+
-
J23113(backboneJ61002)
+
-
Three test tubes of 4 mL LB medium with ampicillin
+
-
37℃ overnight
+
-
==August 2==
+
-
==August 3==
+
-
====Restriction Enzyme processing====
+
-
{|class="wikitable"
+
-
!pSB4K5||BSA|| 10×H Buffer||EcoRI||PstI||MilliQ||Total
+
-
|-
+
-
|8.0||0.5||3.0||0.5||0.5||17.5||30.0
+
-
|}
+
-
{|class="wikitable"
+
-
!B0034||BSA||10×H Buffer||SpeI||PstI||MilliQ||Total
+
-
|-
+
-
|12.0||0.5||3.0||0.5||0.5||13.5||30.0
+
-
|}37℃ 2h
+
-
====DNA purification====
+
-
====Ligation====
+
-
{|class="wikitable"
+
-
!lacP+kil+DT||pSB4K5||ligation high||Total
+
-
|-
+
-
|15||15||15||45
+
-
|}16℃ 1h
+
-
====Miniprep====
+
-
*J23113(backbone J61002) 218.0μg/mL
+
-
*J23113(backbone J61002) 252.5μg/mL
+
-
*J23113(backbone J61002) 201.0μg/mL
+
-
*pSB3C5                  45.0μg/ml  1.60  260/280  1.80  260/230
+
-
*pSB4C5                  213.0μg/mL  1.77  260/280  4.40  260/230
+
-
==August 4==
+
-
==August 5==
+
-
==August 6==
+
-
==August 7==
+
-
==August 8==
+
-
==August 9==
+
-
==August 10==
+
-
====Ligation====
+
-
{|class="wikitable"
+
-
!B0034 SpeI PstI||GFP+DT XbaI PstI||ligation high||Total
+
-
|-
+
-
|2||3||4||9
+
-
|}16℃ 1h
+
-
 
+
-
====Transformation====
+
-
*RBS+GFP+DT
+
-
*lacP+kil+DT
+
-
*RBS（for control）
+
-
:To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
+
-
:Heat shock for 60s at 42℃ 
+
-
:To leave on ice for 2min
+
-
:To spread on ampicillin LB plate
+
-
==August 11==
+
-
==August 12==
+
-
==August 13==
+
-
====Liquid culture====
+
-
B0034 3mL ×3 　　　
+
-
:37℃　overnight
+
-
==August 14==
+
-
====Miniprep====
+
-
:B0034 0μg/mL
+
-
:B0034 235.5μg/mL
+
-
:B0034 76.5μg/mL
+
-
====Colony PCR====
+
-
lacP+kil+DT ×5
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VF||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}25cycles
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f-p||Primer PsPA  r-s||KOD-Plus-||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
 
+
-
====Electrophoresis====
+
-
写真貼ってください
+
-
=====positive control(GFP+DT)=====
+
-
=====negative control(MilliQ)=====
+
-
=====① ～⑥ colony PCR lacP+kil+DT=====
+
-
 
+
-
次の写真
+
-
=====① 　　　1kb ladder =====
+
-
 
+
-
=====② 　　　Positive control (GFP+DT)=====
+
-
=====③ 　　　Negative control (MilliQ)=====
+
-
=====④ ～⑨　lacP+kil+DT　①～⑥=====
+
-
=====⑩～⑫　  pspA 10μL ,6×buffer 2μL=====
+
-
=====⑬　　　　1kb ladder=====
+
-
 
+
-
==August 15==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!2×Quick Taq||pspA r-s||pspA f-p||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
56℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→35cycles<br><br>
+
-
==August 16==
+
-
==August 17==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD-Plus-||MilliQ||Total
+
-
|-
+
-
|5||3||3||1.5||1.5||1||35||50
+
-
|-
+
-
|5||3||5||1.5||1.5||1||33||50
+
-
|}
+
-
negative control(MilliQ 50μL)
+
-
 
+
-
94℃, 2min<br>
+
-
94℃, 15sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→35cycles<br><br>
+
-
 
+
-
====Electrophoresis====
+
-
写真貼ってください
+
-
=====①1kb ladder=====
+
-
 
+
-
=====②, ③　　MgSO4 3μL, pspA=====
+
-
=====④, ⑤　　MgSO4 5μL, pspA=====
+
-
=====⑥      negative control(MilliQ)=====
+
-
=====⑧      1kb ladder=====
+
-
==August 18==
+
-
==August 19==
+
-
==August 20==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!J23113(201.0ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||1||1||3||0.5||14.5||30
+
-
|-
+
-
|}
+
-
==August 21==
+
-
====Colony PCR====
+
-
pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||3||5||1.5||1.5||1||33||50
+
-
|-
+
-
|5||3||7||1.5||1.5||1||31||50
+
-
|-
+
-
|5||3||10||1.5||1.5||1||28||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→25cycles<br><br>
+
-
 
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!J23113(218ng/μL)||Spe1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||1||1||3||0.5||14.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!torA-GFP-DT(20ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.6||0.6||3||0.5||20.3||30
+
-
|}
+
-
====Electrophoresis====
+
-
写真貼ってください
+
-
=====① 　　　1kb ladder=====
+
-
=====②, ③　　torA-GFP-DT(XbaI,PstI)=====
+
-
 
+
-
=====④, ⑤　　J23113(SpeI,PstI)=====
+
-
====Transformation====
+
-
*lacP-torA-GFP-DT(Backbone pSB3C5)
+
-
*BBa_K11704(Backbone pSB1A2)
+
-
====Colony PCR====
+
-
*pspA
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f||Primer PsPA r||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f-p||Primer PsPA r-s||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
*negative control for pspA
+
-
We did PCR without a template.<br>
+
-
*GFP-DT
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
 
+
-
====Transformation====
+
-
*Lacp-torA-GFP-DT(Backbone pSB3C5)
+
-
*J23107-tatABCD
+
-
====PCR====
+
-
kil, torA-GFP
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
→25cycles<br><br>
+
-
==August 22==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|13.3||0.5||0.5||3||0.5||12.2||30
+
-
|}
+
====Gel extraction====
====Gel extraction====
-
pSB3C5(EcoR1, Pst1)<br>
 
-
27.2μg/mL
 
-
 
-
====Transformation(the second time)====
 
-
*Lacp-torA-GFP-DT(Backbone pSB3C5)
 
-
*J23107-tatABCD
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
See "August 21 -Colony PCR-"<br>
 
-
1. 1kb ladder<br>
 
-
2. pspA(Primer pspA f&r)<br>
 
-
3. negative control for 2<br>
 
-
4. GFP-DT<br>
 
-
5. pspA(Primer pspA f-p&r-s)<br>
 
-
6. pspA(Primer pspA f-p&r-s)<br>
 
-
7. negative control for 5&6<br>
 
-
 
-
====PCR====
 
-
Lacp-GFP-DT(a kit of biological parts)
 
-
{|class="wikitable"
 
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 
-
|-
 
-
|5||5||3||1.5||1.5||1||32||1||50
 
-
|}
 
-
====Colony PCR====
 
-
pspA
 
-
{|class="wikitable"
 
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f||Primer pspA r||KOD Plus Neo||MilliQ||Total
 
-
|-
 
-
|5||5||3||1.5||1.5||1||33||50
 
-
|}
 
-
{|class="wikitable"
 
-
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f-p||Primer pspA r-s||KOD Plus Neo||MilliQ||Total
 
-
|-
 
-
|5||5||3||1.5||1.5||1||33||50
 
-
|}
 
-
94℃, 2min<br>
 
-
98℃, 10sec<br>
 
-
60℃, 30sec<br>
 
-
68℃, 30sec<br>
 
-
→30cycles<br>
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
1. 1kb ladder<br>
 
-
2. pspA(Primer pspA f&r)<br>
 
-
3. pspA(Primer pspA f-p&r-s)<br>
 
-
4. negative control for pspA(Primer pspA f&r)<br>
 
-
5. negative control for pspA(Primer pspA f-p&r-s)<br>
 
-
====Transformation====
 
-
Kil
 
-
==August 23==
 
-
====Colony PCR====
 
-
J23107-tatABCD<br>
 
-
VF and VR were used for amplifying approximately 2800bp
 
-
{|class="wikitable"
 
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 
-
|-
 
-
|5||5||3||1.5||1.5||1||32.5||0.5||50
 
-
|}
 
-
94℃, 2min<br>
 
-
98℃, 10sec<br>
 
-
60℃, 30sec<br>
 
-
68℃, 90sec<br>
 
-
→25cycles<br>
 
-
 
-
==August 24==
 
-
====Purification of PCR products====
 
-
pspA(Primer pspA f-p&r-s)<br>
 
-
84.1μg/mL<br>
 
-
→See "August 22 -Colony PCR-"
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!pspA||EcoR1||Pst1||BufferH||BSA||MilliQ||total
 
-
|-
 
-
|20||0.5||0.5||3||0.5||5.5||30
 
-
|}
 
-
{| class="wikitable"
 
-
!pspA||Xba1||Pst1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|20||0.5||0.5||3||0.5||5.5||30
 
-
|}
 
-
at 37℃ for 1h
 
-
====Gel extraction====
 
-
*pspA(EcoR1, Pst1) 30.7μg/mL
 
-
*pspA(Xba1, Pst1) 44.0μg/mL
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!pSB1C3(82.9μg/mL)||EcoR1||Pst1||BufferH||BSA||MilliQ||total
 
-
|-
 
-
|20||0.5||0.5||3||0.5||5.5||30
 
-
|}
 
-
→Gel extraction
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
1. 1kb ladder<br>
 
-
2. J23107-tatABCD →See "August 23 -PCR-"<br>
 
-
3. negative control for J23107-tatABCD<br>
 
-
4. pSB1C3(EcoR1, Pst1)<br>
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!pSB3C5(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||total
 
-
|-
 
-
|1||5||3||9
 
-
|}
 
-
16℃, overnight
 
-
==August 25==
 
-
==August 26==
 
-
==August 27==
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!pSB1C3(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||total
 
-
|-
 
-
|1||5||3||9
 
-
|}
 
-
16℃, overnight
 
-
====Colony PCR====
 
-
Kil (1-7)
 
-
{|class="wikitable"
 
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
 
-
|-
 
-
|5||5||3||1.5||1.5||1||33||50
 
-
|}
 
-
94℃, 2min<br>
 
-
98℃, 10sec<br>
 
-
60℃, 30sec<br>
 
-
68℃, 30sec<br>
 
-
→30cycles<br>
 
写真お願いします。
写真お願いします。
-
 
+
*LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)
-
====Purification of PCR products====
+
-
torA-GFP →See "August 21 -PCR-"
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!Lacp(Backbone:pSB3C5)||Spe1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h<br>
+
-
→Gel extraction<br>
+
-
{| class="wikitable"
+
-
!torA-GFP||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h<br>
+
-
→Purification
+
-
====Purification of PCR products====
+
-
J23107-tatABCD<br>
+
-
42.0μg/mL<br>
+
-
→See "August 23 -PCR-"
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||Spe1||EcoR1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
==August 28==
+
-
====Colony PCR====
+
-
Kil (1-16)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
写真お願いします。
+
-
====Transformation====
+
-
*pspA-pSB1C3
+
-
*pspA-pSB3C5
+
-
====Liquid culture====
+
-
*Kil-3 →Miniprep 100.4μg/mL
+
-
*Kil-6
+
-
*Kil-7 →Miniprep 77.7μg/mL
+
-
*Lacp-torA-GFP-1 →Miniprep 88.3μg/mL
+
-
*Lacp-torA-GFP-2 →Miniprep 85.0μg/mL
+
-
*Lacp-torA-GFP-3 →Miniprep +++
+
-
==August 29==
+
-
====Kil assay====
+
-
evernoteに結果のグラフがあります。
+
-
====Liquid culture====
+
-
*pspA-pSB1C3(1-3)
+
-
*pspA-pSB3C5(1-3)
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!Lacp-Kil-DT(134.7ng/μL)||EcoR1||Spe1||BufferM||MilliQ||total
+
-
|-
+
-
|10||1||1||3||15||30
+
-
|}
+
-
37℃, overnight
+
-
====Colony PCR====
+
-
*Lacp-torA-GFP-DT (1-5)
+
-
*pspA-pSB1C3 (1-5)
+
-
*pspA-pSB3C5 (1-6)
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 40sec<br>
+
-
→30cycles<br>
+
-
====Gel extraction====
+
-
J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL
+
-
==August 30==
+
====Ligation====
====Ligation====
{| class="wikitable"
{| class="wikitable"
-
!J23107-tatABCD(EcoR1, Spe1)22.2μg/mL||DT(EcoR1, Xba1)37.5μg/mL||Ligation High Ver.2||total
+
!J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL||LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||total
|-
|-
-
|10||1||9||20
+
|8||1.5||9.5||19
|}
|}
-
at 16℃ for 1h
 
-
====Transformation====
 
-
J23107-tatABCD-DT (Amp resistance)
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
1. Lacp-torA-GFP-DT-1<br>
 
-
2. Lacp-torA-GFP-DT-2<br>
 
-
3. Lacp-torA-GFP-DT-3<br>
 
-
4. Lacp-torA-GFP-DT-4<br>
 
-
5. Lacp-torA-GFP-DT-5<br>
 
-
6. pspA-pSB1C3-1<br>
 
-
7. pspA-pSB1C3-2<br>
 
-
8. pspA-pSB1C3-3<br>
 
-
9. pspA-pSB1C3-4<br>
 
-
10.pspA-pSB1C3-5<br>
 
-
11.pspA-pSB3C5-1<br>
 
-
12.pspA-pSB3C5-2<br>
 
-
13.pspA-pSB3C5-3<br>
 
-
14.pspA-pSB3C5-4<br>
 
-
15.pspA-pSB3C5-5<br>
 
-
16.pspA-pSB3C5-6<br>
 
-
====Miniprep====
 
-
*pspA-pSB3C5-1 176μg/mL
 
-
*pspA-pSB3C5-2 214μg/mL
 
-
*pspA-pSB3C5-3 461μg/mL
 
-
*pspA-pSB1C3-2 79μg/mL
 
-
====Colony PCR====
 
-
Lacp-torA-GFP-DT (6-13)
 
-
{|class="wikitable"
 
-
!2×Quick Taq||VF||VR||MilliQ||Total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
94℃, 2min<br>
 
-
94℃, 30sec<br>
 
-
56℃, 30sec<br>
 
-
68℃, 1min<br>
 
-
→30cycles<br><br>
 
-
====Restriction====
 
{| class="wikitable"
{| class="wikitable"
-
!pspA-pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
!LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||total
|-
|-
-
|15||0.5||0.5||5||0.5||28.5||50
+
|1.5||1.5||3
|}
|}
-
at 37℃ for 1h
+
at 4℃ for 1 hour
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
3. pspA-pSB1C3(EcoR1, Spe1)<br>
+
-
==August 31==
+
-
====Gel extraction====
+
-
写真お願いします。<br>
+
-
pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL
+
-
====Purification====
+
-
Lacp-GFP-DT -0.7μg/mL
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||total
+
-
|-
+
-
|25||1||13||39
+
-
|}
+
-
at 16℃ for 1h
+
====Transformation====
====Transformation====
-
pspA-DT (Amp resistance)
+
*J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
-
==September 1==
+
*LacP-torA-GFP-DT(EcoR1, Xba1)
-
==September 2==
+
-
====Colony PCR====
+
-
J23107-tatABCD-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
 
+
-
pspA-DT (2-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
Extension, 10sec<br>
+
-
→25cycles<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!LacP-torA-GFP-DT||EcoR1||Spe1||BufferH||MilliQ||total
+
-
|-
+
-
|10||1||1||2||6||20
+
-
|}
+
-
at 4℃ for overnight
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
3. J23107-tatABCD-DT-1<br>
+
-
4. J23107-tatABCD-DT-3<br>
+
-
5. J23107-tatABCD-DT-4<br>
+
-
6. J23107-tatABCD-DT-5<br>
+
-
7. J23107-tatABCD-DT-6<br>
+
-
8. J23107-tatABCD-DT-7<br>
+
-
9. J23107-tatABCD-DT-8<br>
+
-
 
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
3. J23107-tatABCD-DT-1<br>
+
-
4. J23107-tatABCD-DT-3<br>
+
-
5. J23107-tatABCD-DT-4<br>
+
-
6. J23107-tatABCD-DT-5<br>
+
-
7. J23107-tatABCD-DT-6<br>
+
-
8. J23107-tatABCD-DT-7<br>
+
-
9. J23107-tatABCD-DT-8<br>
+
-
11. LacP-pSB3C5(SpeI, PstI)
+
-
 
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
3. pspA-DT-2<br>
+
-
4. pspA-DT-3<br>
+
-
5. pspA-DT-4<br>
+
-
6. pspA-DT-5<br>
+
-
7. pspA-DT-6<br>
+
-
8. pspA-DT-7<br>
+
-
9. pspA-DT-8<br>
+
-
 
+
-
 
+
-
==September 3==
+
-
====PCR====
+
-
tatABCD-DT
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
====Gel extraction====
+
-
写真お願いします。<br>
+
-
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA-GFP-DT||XbaI||PstI||BufferM||MilliQ||total
+
-
|-
+
-
|20||2||2||4||12||40
+
-
|}
+
-
====Purification====
+
-
J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
2. J23107-tatABCD<br>
+
-
====Liquid culture====
+
-
*LacP-kil-DT(1)
+
-
*pspA-DT(4, 7)
+
-
====PCR====
+
-
J23107-tatABCD
+
-
{|class="wikitable"
+
-
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||29||4||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 10sec<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
写真お願いします。<br>
+
-
LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230)
+
-
LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)
+
-
====Transformation====
+
-
J23107-tatABCD (Amp resistance)<br>
+
-
J23113-kil-DT (Amp resistance)<br>
+
-
J23100-kil-DT (CP resistance)<br>
+
-
J23101-kil-DT (CP resistance)<br>
+
-
J23106-kil-DT (CP resistance)<br>
+
-
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
+
-
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
+
-
 
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1. 1kb ladder<br>
+
-
2-7. LacP-torA-GFP-DT<br>
+
-
====Colony PCR====
+
-
LacP-torA-GFP-DT (14-21)
+
-
J23113-kil-DT (Amp resistance, 1-4)
+
-
J23113-kil-DT (CP resistance, 1-4)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||primer-f||primer-r||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→25cycles<br><br>
+
-
==September 4==
+
-
====Liquid culture====
+
-
*LacP-kil-DT(4, 5)(Amp resistance)
+
-
*pspA-DT(3, 6, 7)(Amp resistance)
+
-
====PCR====
+
-
We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1-8. Lacp-torA-GFP-DT<br>
+
-
 
+
-
写真お願いします。<br>
+
-
1-4. J23113-kil-DT (Amp resistance)<br>
+
-
5-8. J23113-kil-DT (CP resistance)<br>
+
-
====Liquid culture====
+
-
*LacP-torA-GFP-DT
+
-
*J23113-kil-DT
+
-
*LacP-kil-DT(with IPTG)
+
-
*LacP-kil-DT(without IPTG)
+
-
 
+
-
After 20 hour, we quantified OD600.
+
-
{|class="wikitable"
+
-
!plasmid||OD600
+
-
|-
+
-
|LacP-kil-DT(with IPTG)||2.116
+
-
|-
+
-
|LacP-kil-DT(without IPTG)||2.320
+
-
|}
+
-
 
+
-
We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours.
+
-
We quantify OD600.
+
-
{|class="wikitable"
+
-
!time||1||2||3
+
-
|-
+
-
|2hours||0.131||0.100||0.606
+
-
|-
+
-
|add an antibiotic||Amp||Kan||none
+
-
|-
+
-
|7hours||0.491||0.988||2.634
+
-
|-
+
-
|19hours||2.253||0.815||2.742
+
-
|}
+
-
==September 5==
+
-
====Colony PCR====
+
-
J23107-tatABCD (1-7)
+
-
{|class="wikitable"
+
-
!buffer||2mM dNTPs||25mM MgSO4||primer-f||primer-r||KOD Plus Neo||DW||Total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.25min<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
<small>by Kato</small><br>
+
-
See "September 3 -Gel extraction-"
+
-
LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230)
+
-
LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)
+
-
====Miniprep====
+
-
*J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
+
-
*LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
+
-
*LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
+
-
*LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
+
-
*pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
+
-
*pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
+
-
*pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pspA-DT-6||XbaI||PstI||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃ for 1h
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
J23107-tatABCD (1-7)<br>
+
-
====Colony PCR====
+
-
J23107-tatABCD (8-23)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Gel extraction====
+
-
pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!pspA-DT(XbaI, PstI)||LacP(pSB3C5)(SpeI, PstI)||Ligation High Ver.2||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 16℃
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
Lacp-torA-GFP-DT(8-15)<br>
+
-
 
+
-
写真お願いします。<br>
+
-
Lacp-torA-GFP-DT(9-23)<br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Lacp-torA-GFP-DT(EcoRI, SpeI)||DT 17.2μg/mL(XbaI, PstI)||Ligation High Ver.2||total
+
-
|-
+
-
|15||25||20||60
+
-
|}
+
-
====Liquid culture====
+
-
*J23107-tatABCD-8
+
-
*J23107-tatABCD-20
+
-
*Lacp-torA-GFP-DT-21
+
-
==September 6==
+
-
====Miniprep====
+
-
*J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
+
-
*J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)
+
-
 
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD 102.9μg/mL||SpeI||PstI||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃ for 1h
+
-
====Transformation====
+
-
LacP-pspA-DT(pSB3C5)
+
-
{|class="wikitable"
+
-
!DNA||Competent cell||total
+
-
|-
+
-
|2||20||22
+
-
|}
+
-
====Gel extraction====
+
-
写真お願いします。<br>
+
-
J23107-tatABCD(SpeI, PstI)
+
-
→We failed gel extraction because we mistook steps of experiment.
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
LacP-torA-GFP-DT-pspA-DT<br>
+
-
====Restriction====
+
-
<small>by Terasaka</small><br>
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||SpeI||PstI||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
====Gel extraction====
+
-
写真お願いします。<br>
+
-
J23107-tatABCD(SpeI, PstI)
+
-
→See "September 7 -Gel extraction-"
+
-
==September 7==
+
-
====Liquid culture====
+
-
*Lacp-torA-GFP-DT-1 (CP resistance)
+
-
*Lacp-torA-GFP-DT-21 (CP resistance)
+
-
preculture for 1 hour
+
-
 
+
-
====Colony PCR====
+
-
Lacp-torA-GFP-DT (1-8)
+
-
Lacp-pspA-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→30cycles<br><br>
+
-
====Restriction====
+
-
<small>by Terasaka</small><br>
+
-
{| class="wikitable"
+
-
!J23107-tatABCD||EcoRI||SpeI||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
{| class="wikitable"
+
-
!GFP-DT||XbaI||PstI||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||0.5||0.5||3||0.5||10.5||30
+
-
|}
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
① -⑧. LacP-pspA-DT<br>
+
-
1-8. LacP-torA-GFP-DT<br>
+
-
====Gel extraction====
+
-
写真お願いします。
+
-
*J23107-tatABCD(EcoR1, Spe1)
+
-
*J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
+
-
*GFP-DT(Xba1, Pst1)
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pspA-DT(145.5μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
37℃, overnight
+
-
====Liquid culture====
+
-
*Lacp-torA-GFP-DT-7 (LB 2mL)<br>
+
-
+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM
+
-
*Lacp-torA-GFP-DT-7 (LB 5mL)
+
-
==September 8==
+
-
====Check the effect of Amp====
+
-
We used 5mL of the liquid culture medium with Lacp-torA-GFP-DT-7.<br>
+
-
→See "September 7 -Liquid culture-"<br>
+
-
We diluted it with LB until OD600=0.444, and dispensed it.<br>
+
-
The dispense volume was 1.3mL.<br>
+
-
We added 0/13/130μL of Amp(50mg/mL) to it.<br>
+
-
=====5.5h after incubating at 37℃=====
+
-
{| class="wikitable"
+
-
!Amp||0μL||13μL||130μL
+
-
|-
+
-
|OD600||2.457||2.696||0.310
+
-
|}
+
====Observation through a confocal microscope====
====Observation through a confocal microscope====
-
We used the liquid culture media with Lacp-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.<br>
+
We made sample for observation through a confocal microscope
-
Their OD600 was 0.47.<br>
+
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.
-
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.<br>
+
Then, we incubated onto medium without IPTG for 2.5 hours.
-
2.5h after incubating at 37℃, we observed them through a confocal microscope.<br>
+
-
We failed to observe it.
+
-
====Gel extraction====
 
-
pspA-DT -5.5μg/mL
 
-
====Restriction====
 
{| class="wikitable"
{| class="wikitable"
-
!pspA-DT-3(322μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
!IPTG(mM)||medium
-
|-
+
-
|10||1||1||3||3||12||30
+
-
|}
+
-
at 37℃ for 1h
+
-
==September 9==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(102.9μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
+
-
|-
+
-
|10||1||1||3||15||30
+
-
|}
+
-
====Electrophoresis====
+
-
写真お願いします。<br>
+
-
1. J23107-tatABCD(EcoR1, Spe1)<br>
+
-
2. J23107-tatABCD(before restriction)<br>
+
-
3. pspA-DT(Xba1, Pst1)<br>
+
-
4. pspA-DT(before restriction)<br>
+
-
====Gel extraction====
+
-
写真(2枚)お願いします。<br>
+
-
*J23107-tatABCD(EcoR1, Spe1) 26μg/mL
+
-
*pspA-DT(Xba1, Pst1) 199μg/mL
+
-
====Liquid culture====
+
-
*J23100/J23101/J23106-1
+
-
*pspA-DT-5 ×2
+
-
*Lacp-torA-GFP-DT-21
+
-
*GFP generator-2
+
-
*Lacp-pspA-DT-6
+
-
==September 10==
+
-
====Miniprep====
+
-
*J23100-1  105.5μg/mL
+
-
*J23101-1  76.0μg/mL
+
-
*J23106-1  90.9μg/mL
+
-
*pspA-DT-5  ①104.1μg/mL  ②80.0μg/mL
+
-
*Lacp-torA-GFP-DT-21  79.6μg/mL
+
-
*GFP generator-2  84.1μg/mL
+
-
*Lacp-pspA-DT-6  99.8μg/mL
+
-
====Ligation====
+
-
*J23107-tatABCD-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||total
+
|-
|-
-
|10||5||7.5||22.5
+
|0||LB
-
|}
+
-
*Negative control for J23107-tatABCD-DT
+
-
{| class="wikitable"
+
-
!DT(EcoR1, Xba1)||Ligation High Ver.2||total
+
|-
|-
-
|5||5||10
+
|0.1||LB
-
|}
+
-
*J23107-tatABCD-pspA-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(Spe1, Pst1)||pspA-DT(Xba1, Pst1)||Ligation High Ver.2||total
+
|-
|-
-
|3||6||9||18
+
|0.1||LB with 0.1mM IPTG
-
|}
+
-
*Negative control for J23107-tatABCD-pspA-DT
+
-
{| class="wikitable"
+
-
!J23107-tatABCD(Spe1, Pst1)||Ligation High Ver.2||total
+
|-
|-
-
|3||3||6
+
|0||LB with 2 percent glucose
-
|}
+
-
====Restriction====
+
-
写真お願いします。
+
-
{| class="wikitable"
+
-
!DNA||Spe1||Pst1||BufferH||MilliQ||total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)<br>
+
-
at 37℃
+
-
====Gel extraction====
+
-
写真（２まい）お願いします。<br>
+
-
J23100  27.1μg/mL<br>
+
-
J23101  50.2μg/mL<br>
+
-
J23106  16.1μg/mL<br>
+
-
====Ligation====
+
-
*J23100/J23101/J23106-kil-DT
+
-
{| class="wikitable"
+
-
!J23100/J23101/J23106(Spe1, Pst1)||kil-DT(Xba1, Pst1)||Ligation High Ver.2||total
+
|-
|-
-
|1||5||3||9
+
|0.1||LB with 2 percent glucose
-
|}
+
-
*Negative control for J23100/J23101/J23106-kil-DT
+
-
{| class="wikitable"
+
-
!J23100/J23101/J23106(Spe1, Pst1)||MilliQ||Ligation High Ver.2||total
+
|-
|-
-
|1||5||3||9
+
|0||LB with 5 percent glucose
-
|}
+
-
====Transformation====
+
-
J23100/J23101/J23106-kil-DT
+
-
==September 11==
+
-
====Colony PCR====
+
-
*J23107-tatABCD-DT (1-8)
+
-
*J23107-tatABCD-pspA-DT (1-8)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF2||VR||MilliQ||Total
+
|-
|-
-
|25||1||1||23||50
+
|0.1||LB with 5 percent glucose
-
|}
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 3.5min<br>
+
-
→30cycles
+
-
====Electrophoresis====
+
-
写真お願いします。
+
-
*J23107-tatABCD-DT (1-8)
+
-
*J23107-tatABCD-pspA-DT (1-8)
+
-
====Restriction====
+
-
写真お願いします。
+
-
{| class="wikitable"
+
-
!GFP generator(84.1μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Lacp-torA-GFP-DT(85.0μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
+
-
|-
+
-
|15||1||1||3||10||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Lacp-pspA-DT(99.8μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||1||1||3||3||7||30
+
-
|}
+
-
====Liquid culture====
+
-
pSB1C3(self-ligation) ×5 in LB 3mL+CP 3μL<br>
+
-
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.<br>
+
-
1h and 5h after incubating at 37℃, we measured OD600.<br>
+
-
{| class="wikitable"
+
-
!||1h||5h
+
-
|-
+
-
|0||-0.020||0.403
+
|-
|-
-
|30||-0.046||0.544
+
|0||LB with 2 percent glucose
|-
|-
-
|60||0.263||0.007
+
|0||LB ここ途中
|-
|-
-
|150||0.061||0.839
+
|0||LB
|-
|-
-
|300||0.093||1.441
+
|0||LB
|}
|}
-
====Ligation====
+
Then, we incubated onto medium without IPTG for 2.5 hours.
-
{| class="wikitable"
+
-
!pSB3C5(EcoR1, Pst1)||pspA-DT(Xba1, Pst1)||J23107-tatABCD(EcoR1, Spe1)||Ligation High Ver.2||total
+
-
|-
+
-
|1||3||5||4.5||13.5
+
-
|}
+
-
4℃, overnight
+
-
====Colony PCR====
+
-
*J23100-kil-DT (1-6)
+
-
*J23101-kil-DT (1-7)
+
-
*J23106-kil-DT (1-2)
+
-
{|class="wikitable"
+
-
!2×Quick Taq||VF||VR||MilliQ||Total
+
-
|-
+
-
|23||1||1||25||50
+
-
|}
+
-
====Gel extraction====
+
-
写真お願いします。
+
-
*GFP generator (Xba1, Pst1) 14.1μg/mL
+
-
*lacp-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
+
-
*lacp-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)
+
-
====Electrophoresis====
+
-
写真お願いします。
+
-
*J23107-tatABCD-DT (1-8)
+
-
*J23106-kil-DT (1, 2)
+
-
*J23100-kil-DT (1-4)
+
-
====Liquid culture====
+
-
*Lacp-torA-GFP-DT
+
-
*pSB1C3
+
-
====Colony PCR====
+
-
We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.
+
-
==September 12==
+
-
====Colony PCR====
+
-
J23107-tatABCD-pspA-DT (9-15)
+
-
94℃, 2min<br>
+
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 4min<br>
+
-
→26cycles<br><br>
+
-
====Check the effect of Amp====
+
-
We cultured pSB1C3 for overnight.<br>
+
-
→See "September 7 -Liquid culture-"<br>
+
-
We diluted it with LB, and dispensed it.<br>
+
-
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .<br>
+
-
{|class="wikitable"
+
-
!Amp||start||5 hours
+
-
|-
+
-
|1/10||0.557||0.077
+
-
|-
+
-
|1/30||0.628||0.166
+
-
|-
+
-
|1/50||0.472||0.055
+
-
|-
+
-
|0||0.594||2.585
+
-
|}
+
-
====Electrophoresis====
+
-
写真お願いします。
+
-
*J23107-tatABCD-pspA-DT
+
-
写真お願いします。
 
-
*J23101-kil-DT (1-7)
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!pspA(3C5) (106μg/mL)||EcoR1||Pst1||BufferH||MilliQ||total
 
-
|-
 
-
|15||1||1||3||10||30
 
-
|}
 
-
{| class="wikitable"
 
-
!pspA-DT-3||Xba1||Pst1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|15||1||1||3||3||7||30
 
-
|}
 
-
{| class="wikitable"
 
-
!J23107-tatABCD (102.4μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
 
-
|-
 
-
|15||1||1||3||10||30
 
-
|}
 
-
====Electrophoresis====
 
-
写真お願いします。
 
-
*pspA(3C5)(EcoR1, Pst1)
 
-
*pspA-DT(Xba1, Pst1)
 
-
*J23107-tatABCD(EcoR1, Spe1)
 
-
*pSB1C3(EcoR1, Spe1)
 
-
====Transformation====
 
-
J23107-tatABCD-pspA-DT(pSB3C5)
 
-
{|class="wikitable"
 
-
!DNA||Competent cell||total
 
-
|-
 
-
|2||20||22
 
-
|}
 
-
on ice for 30 minites
 
-
heatshock at 42℃ for 1 hour
 
-
on ice 2 minites
 
-
then add SOC medium 200μl
 
-
preculture at 37℃ for 1 hour
 
-
incubate CP culture plate
 
-
====Gel extraction====
 
-
写真お願いします。
 
-
*pspA(3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
 
-
*pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
 
-
*J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||pspA-DT(Xba1, Pst1) 26.1μg/mL||J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL||Ligation High Ver.2||total
 
-
|-
 
-
|1||3||6||5||15
 
-
|}
 
-
{| class="wikitable"
 
-
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||MilliQ||Ligation High Ver.2||total
 
-
|-
 
-
|1||9||5||15
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||GFP-DT(Xba1, Pst1) 7.0μg/mL||Ligation High Ver.2||total
 
-
|-
 
-
|1||5||3||9
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||MilliQ||Ligation High Ver.2||total
 
-
|-
 
-
|1||5||3||9
 
-
|}
 
-
4℃, overnight
 
-
====Observation through a confocal microscope====
 
-
Evernoteに上がっている写真をお願いします。<br>
 
-
==September 13==
 
-
====Miniprep====
 
-
*J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
 
-
|-
 
-
|10||1||1||3||15||30
 
-
|}
 
-
====Transformation====
 
-
*LacP-GFP generater
 
-
*LacP
 
-
*J23107-tatABCD-pspA-DT(pSB3C5)
 
-
*J23107-tatABCD
 
-
====Colony PCR====
 
-
J23107-tatABCD-pspA-DT<br>
 
-
→See "September 14 -Colony PCR-"
 
-
==September 14==
 
-
====Colony PCR====
 
-
写真お願いします。<br>
 
-
See "September 13 -Colony PCR-"<br>
 
-
J23107-tatABCD-pspA-DT<br>
 
-
====Colony PCR====
 
-
*J23107-tatABCD-pspA-DT (1-6)
 
-
{|class="wikitable"
 
-
!2×Quick Taq||VF||VR||MilliQ||Total
 
-
|-
 
-
|23||1||1||25||50
 
-
|}
 
-
94℃, 2min<br>
 
-
94℃, 30sec<br>
 
-
55℃, 30sec<br>
 
-
68℃, 4min<br>
 
-
→30cycles<br><br>
 
-
==September 15==
 
-
====Liquid culture====
 
-
*J23107-tatABCD-pspA-DT-1
 
-
*J23107-tatABCD-pspA-DT-2
 
-
*J23107-tatABCD-pspA-DT-3
 
-
====Miniprep====
 
-
*J23107-tatABCD-pspA-DT-1 22.9μg/mL
 
-
*J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
 
-
*J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
 
-
|-
 
-
|15||0.5||0.5||3||11||30
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT (79.6μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|10||0.5||0.5||3||0.5||15.5||30
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP-kil-DT (56.0μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|15||0.5||0.5||3||0.5||10.5||30
 
-
|}
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Pst1||BufferM||MilliQ||total
 
-
|-
 
-
|15||0.5||0.5||3||11||30
 
-
|}
 
-
37℃, 2 hours
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
①. 1kb ladder<br>
 
-
②. J23107-tatABCD-pspA-DT<br>
 
-
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 
-
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 
-
⑤. LacP-kil-DT<br>
 
-
⑥. LacP-kil-DT(EcoR1, Xba1)<br>
 
-
⑦. LacP-torA-GFP-DT<br>
 
-
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
 
-
====Gel extraction====
 
-
写真お願いします。<br>
 
-
①. 1kb ladder<br>
 
-
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 
-
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 
-
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 
-
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 
-
⑥. 1kb ladder<br>
 
-
*J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
 
-
*J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)
 
-
====Purification====
 
-
*LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
 
-
*LacP-kil-DT(EcoR1, Xba1) →failed
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT(EcoR1, Xba1)||J23107-tatABCD-pspA-DT(EcoR1, Spe1)||Ligation High Ver.2||total
 
-
|-
 
-
|4||10||7||21
 
-
|}
 
-
{| class="wikitable"
 
-
!pSB1C3(EcoR1, Pst1)||J23107-tatABCD-pspA-DT(EcoR1, Pst1)||Ligation High Ver.2||total
 
-
|-
 
-
|4||10||7||21
 
-
|}
 
-
at 16℃ for overnight
 
-
 
-
==September 16==
 
-
====Transformation====
 
-
LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT<br>
 
-
J23107-tatABCD-pspA-DT(pSB1C3)<br>
 
-
 
-
==September 17==
 
-
====Colony PCR====
 
-
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
 
-
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 
-
J23107-kil-DT (6-10)
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT (126μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|10||1||1||3||3||12||30
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT (126μg/mL)||Xba1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|3||1||3||3||20||30
 
-
|}
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
 
-
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 
-
====Liquid culture====
 
-
*J23107-tatABCD-pspA-DT(pSB1C3)
 
-
*J23107-tatABCD-pspA-DT(pSB3C5)
 
-
*LacP-torA-GFP-DT(pSB3C5)
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
lane1. 1kb ladder<br>
 
-
lane2. J23101-kil-DT-6<br>
 
-
lane3. J23101-kil-DT-7<br>
 
-
lane4. J23101-kil-DT-8<br>
 
-
lane5. J23101-kil-DT-10<br>
 
-
lane6. LacP-torA-GFP-DT<br>
 
-
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
 
-
lane8. LacP-torA-GFP-DT(Xba1)<br>
 
-
====Miniprep====
 
-
*J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)
 
-
 
-
==September 18==
 
====Restriction enzyme processing====  
====Restriction enzyme processing====  
Line 3,043: Line 75:
|}<br>
|}<br>
37℃<br>
37℃<br>
-
 
-
==September 19==
 
-
 
-
====GeneClean II====
 
-
LacP-torA-GFP-DT<br>
 
-
4k5
 
-
====Ligation====
 
-
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT(X,P) 9.7μg/mL||pSB4K5(E,P) 9.6μg/mL||J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL||Ligation High Ver.2||total
 
-
|-
 
-
|7||2||7||8||24μL
 
-
|}
 
-
 
-
{| class="wikitable"
 
-
!pSB4K5(E,P) 9.6μg/mL||Ligation High Ver.2||MilliQ||total
 
-
|-
 
-
|2||1||21||24μL
 
-
|}<br>
 
-
 
-
incubate at 37℃ for 2h
 
-
 
-
====Electrophoresis====
 
-
 
-
J23101-kil-DT(X,P) (X) (P)<br>
 
-
Lane1: 1kb ladder<br>
 
-
Lane2: empty<br>
 
-
Lane3: plasmid<br>
 
-
Lane4: X<br>
 
-
Lane5: P<br>
 
-
Lane6: X,P<br>
 
-
 
-
====Liquid Culture====
 
-
 
-
We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB medium+Amp.
 
-
 
-
====Transformation====
 
-
 
-
J2-tat-psp-DT-LacP-torAGFP-DT<br>
 
-
pSB4K5 negative control<br>
 
-
 
-
====Liquid culture====
 
-
J2-tat-psp-DT-LacP-torAGFP-DT:1~5<br>
 
-
 
-
====Colony PCR====
 
-
J2-tat-psp-DT-LacP-torAGFP-DT<br>
 
-
{|class="wikitable"
 
-
!VF2||VR||Quick tag||MilliQ||total
 
-
|-
 
-
|1||1||25||23||50
 
-
|}
 
-
 
-
Predenature 94℃  2min<br>
 
-
Denature    94℃  30sec<br>
 
-
Annealing  55℃ 30sec<br>
 
-
Extension  68℃  5min<br>
 
-
→30cycles<br><br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT(4) 62.5μg/mL||XbaI||PstI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|10||1||1||3||12||3||30
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT(4)||EcoRI||PstI||MilliQ||BufferH||total
 
-
|-
 
-
|10||1||1||15||3||30
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT||XbaI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|2||0.5||1||5.5||1||10
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT||EcoRI||MilliQ||BufferH||total
 
-
|-
 
-
|2||0.5||6.5||1||10
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT||PstI||MilliQ||BufferH||total
 
-
|-
 
-
|2||0.5||6.5||1||10
 
-
|}
 
-
 
-
at 37℃, 2 hours
 
-
 
-
====Electrophoresis====
 
-
 
-
Lane1: 1kb ladder<br>
 
-
Lane2: empty<br>
 
-
Lane3: plasmid<br>
 
-
Lane4: E<br>
 
-
Lane5: X<br>
 
-
Lane6: P<br>
 
-
Lane7: E,P<br>
 
-
Lane8: X,P<br>
 
-
 
-
====Electrophoresis====
 
-
 
-
J2-tat-psp-DTLacP-torAGFP-DT<br>
 
-
 
-
Lane1: ladder<br>
 
-
Lane2: 1<br>
 
-
Lane3: 2<br>
 
-
Lane4: 3<br>
 
-
Lane5: 4<br>
 
-
Lane6: 5<br>
 
-
 
-
====Ligation====
 
-
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT(X,P) 9.7μg/mL||pSB4K5(E,P) 9.6μg/mL||J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL||Ligation High Ver.2||total
 
-
|-
 
-
|7||4||7||9||27μL
 
-
|}<br>
 
-
 
-
(Negative control)<br>
 
-
{| class="wikitable"
 
-
!pSB4K5(E,P) 9.6μg/mL||Ligation High Ver.2||MilliQ||total
 
-
|-
 
-
|4||9||14||27μL
 
-
|}<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoRI||Pst||total
 
-
|-
 
-
|10||3||15||1||1||30
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoRI||Pst||total
 
-
|-
 
-
|3||1||5.5||0.5||0||10
 
-
|}
 
-
 
-
{|class="wikitable"
 
-
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoRI||Pst||total
 
-
|-
 
-
|3||1||5.5||0||0.5||10
 
-
|}
 
-
 
-
====PCR====
 
-
{|class="wikitable"
 
-
!torA-R9||MilliQ||10xPCR buffer for KOD plus neo||2mM dNTP3||25mM MgSO4||M13||KOD plus neo||total
 
-
|-
 
-
|0.5||34||5||5||3||1.5||1||50
 
-
|}
 
-
 
-
Predenature 94℃  2min<br>
 
-
Denature    98℃  10sec<br>
 
-
Annealing  60℃ 30sec<br>
 
-
Extension  68℃ 20sec<br>
 
-
→35cycles<br><br>
 
-
 
-
====Electrophoresis====
 
-
 
-
torA-R9,plasmid,(E),(P),(E,P)<br>
 
-
 
-
Lane1: 1kb ladder<br>
 
-
Lane2: empty<br>
 
-
Lane3: plasmid<br>
 
-
Lane4: E<br>
 
-
Lane5: P<br>
 
-
Lane6: E,P<br>
 
-
Lane7: empty<br>
 
-
Lane8: 1kb ladder<br>
 
-
 
-
====Liquid culture====
 
-
 
-
LacP-tatA9FT-DT (9/4)
 
-
 
-
====PCR purification====
 
-
 
-
torA-R9 68.0μg/mL
 
-
 
-
==September 20==
 
-
 
-
 
-
 
-
====Gel extraction====
 
-
LacP-kil-DT (E,P) (X,P)<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!torA-R9 68.0μg/mL||bufferM||SpeI||MilliQ||Total
 
-
|-
 
-
|20||3||1||6||30
 
-
|}
 
-
at 37℃, 2h
 
-
 
-
====Ligation====
 
-
 
-
{| class="wikitable"
 
-
!LacP-kil-DT(X,P) 12.4μg/mL||pSB1C3(E,P) 34.2μg/mL||J23107-tatABCD-PsPA-DT(E,S) 14.2μg/mL||Ligation High Ver.2||total
 
-
|-
 
-
|7||4||7||9||27μL
 
-
|} 16℃, 1h<br>
 
-
 
-
(Negative control)<br>
 
-
{| class="wikitable"
 
-
!pSB1C3(E,P) ||Ligation High Ver.2||MilliQ||total
 
-
|-
 
-
|4||9||14||27μL
 
-
|}<br>
 
-
 
-
====Transformation====
 
-
 
-
Using above Ligation and Negative control's product and torA-R9.<br>
 
-
 
-
====Miniprep====
 
-
 
-
J23106-kil-DT 6;  12.0μg/mL, (260/280): 1.78, (260/230): 1.73<br>
 
-
J23106-kil-DT 7;  13.6μg/mL, (260/280): 1.85, (260/230): 1.73<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!GFP-DT 84.1μg/mL||bufferM||10xBSA||BbaI||MilliQ||total
 
-
|-
 
-
|15||3||3||1||8||30
 
-
|}
 
-
at 37℃, 2 hours
 
-
 
-
====Colony PCR====
 
-
 
-
We made J2-tat-psp-DT-LacP-torAGFP-DT's 1~8 samples.
 
-
{|class="wikitable"
 
-
!Quick tag||tat f primer||VR||MilliQ||total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
 
-
Predenature 94℃  2min<br>
 
-
Denature    94℃  30sec<br>
 
-
Annealing  54℃ 30sec<br>
 
-
Extension  68℃ 4min<br>
 
-
→30cycles<br>
 
-
 
-
 
-
====Liquid culture====
 
-
 
-
We did liquid culture  J2-tat-psp-DT-LacP-torAGFP-DT's 1~4 samples.
 
-
 
-
====Electrophoresis====
 
-
 
-
J2-tat-psp-DT-LacP-torAGFP-DT
 
-
 
-
Lane1: 1kb ladder<br>
 
-
Lane2: 1<br>
 
-
Lane3: 2<br>
 
-
Lane4: 3<br>
 
-
Lane5: 4<br>
 
-
Lane6: 5<br>
 
-
Lane7: 6<br>
 
-
Lane8: 7<br>
 
-
Lane9: 8<br>
 
-
Lane10: 1kb ladder<br>
 
-
Lane11: empty<br>
 
-
Lane12: empty<br>
 
-
 
-
torA-R9 (E,P): 31μg/mL, (280/280): 0.80, (280/230): 0.39<br>
 
-
1C3(E,P): 22μg/mL, (280/280):1.33, (280/230):0.6<br>
 
-
torA-R9 (S): 56μg/mL, (280/280): 1.11, (280/230): 0.69<br>
 
-
GFP-DT(X): 45μg/mL, (280/280): 1.15, (280/230): 0.88<br>
 
-
 
-
====Ligation====
 
-
 
-
{| class="wikitable"
 
-
!torA-R9 (E,P)||1C3(E,P)||Ligation High Ver.2||total
 
-
|-
 
-
|8||2||5||15μL
 
-
|}
 
-
{| class="wikitable"
 
-
!torA-R9 (S)||1C3(X)||Ligation High Ver.2||total
 
-
|-
 
-
|3||3||3||9μL
 
-
|}
 
-
at 16℃, 1hour
 
-
 
-
====Colony PCR====
 
-
 
-
J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)<br>
 
-
numbering 9~15
 
-
 
-
====Transformation====
 
-
J23107-tat-psp-DT-lacP-torA-GFP-DT (4k5)<br>
 
-
J23107-tat-psp-DT-lacP-torA-kil-DT(1C3) <br>
 
-
Negative control (1C3)<br>
 
-
Negative control(4K5)<br>
 
-
torA-R9(1C3)<br>
 
-
 
-
====Liquid culture====
 
-
 
-
J23107-tat-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)<br>
 
-
{|class="wikitable"
 
-
!2xQuick tag||VF||VR||MilliQ||total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
 
-
Predenature 94℃  2min<br>
 
-
Denature    94℃  30sec<br>
 
-
Annealing  55℃ 30sec<br>
 
-
Extension  68℃ 5min<br>
 
-
→30cycles<br>
 
-
 
-
====PCR====
 
-
 
-
{|class="wikitable"
 
-
!torA-R9-GFP-DT||MilliQ||KOD plus ver.2 buffer||dNTP3||MgSO4||M13||VR||KOD plus||total
 
-
|-
 
-
|1||35||5||5||2||1.5||1.5||1||52
 
-
|}
 
-
 
-
Predenature 94℃  2min<br>
 
-
Denature    94℃  15sec<br>
 
-
Annealing  55℃ 30sec<br>
 
-
Extension  68℃ 5min<br>
 
-
→30cycles<br>
 
-
 
-
====Miniprep====
 
-
 
-
J2-tat-psp-DT-LacP-torA-GFP-DT:2,3<br>
 
-
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95<br>
 
-
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79<br>
 
-
(*)100μL
 
-
 
-
=====Electrophoresis=====
 
-
 
-
torA-R9-GFP-DT(after PCR)
 
-
 
-
==September 21==
 
-
 
-
====Gel extraction====
 
-
 
-
torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49<br>
 
-
 
-
====Electrophoresis====
 
-
 
-
J23107-tat-psp-DT-LacP-torA-GFP-DT(kan): 9~15<br>
 
-
 
-
====Electrophoresis====
 
-
 
-
J2-tat-psp-DT-LacP-torA-GFP-DT: 1,2<br>
 
-
 
-
Lane1: empty<br>
 
-
Lane2: empty<br>
 
-
Lane3: 2<br>
 
-
Lane4: 3<br>
 
-
Lane5: 1kb ladder<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!LacP-kil-DT(3)||XbaI||PstI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|15||1||1||3||7||3||30
 
-
|}
 
-
{|class="wikitable"
 
-
!LacP-torA-6FP-DP(9/5)||XbaI||PstI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|15||1||1||3||7||3||30
 
-
|}
 
-
{|class="wikitable"
 
-
!LacP-kil-DT||XbaI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|2||0.5||1||5.5||1||10
 
-
|}
 
-
{|class="wikitable"
 
-
!LacP-torA-6FP-DP||XbaI||10xBSA||MilliQ||BufferM||total
 
-
|-
 
-
|2||0.5||1||5.5||1||10
 
-
|}
 
-
{|class="wikitable"
 
-
!LacP-kil-DT||PstI||MilliQ||BufferH||total
 
-
|-
 
-
|2||0.5||6.5||1||10
 
-
|}
 
-
{|class="wikitable"
 
-
!LacP-torA-6FP-DP||XbaI||PstI||MilliQ||BufferH||total
 
-
|-
 
-
|2||0.5||1||6.5||1||10
 
-
|}
 
-
{|class="wikitable"
 
-
!J2-tat-psp-DT||SpeI||PstI||BufferH||MilliQ||total
 
-
|-
 
-
|20||1||1||4||14||40
 
-
|}
 
-
{|class="wikitable"
 
-
!J2-tat-psp-DT||SpeI||BufferM||MilliQ||total
 
-
|-
 
-
|2||0.5||1||6.5||10
 
-
|}
 
-
{|class="wikitable"
 
-
!J2-tat-psp-DT||PstI||BufferH||MilliQ||total
 
-
|-
 
-
|2||0.5||1||6.5||10
 
-
|}<br>
 
-
at 37℃, 2hours
 
-
 
-
==September 22==
 
-
 
-
====Miniprep====
 
-
torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42<br>
 
-
torA-R9(Amp)2:  97.8μg/mL, (260/280): 1.67, (280/230): 1.72<br>
 
-
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67<br>
 
-
LacP-torAGFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27<br>
 
-
 
-
====Electrophoresis====
 
-
 
-
LaneA: J2-tat (9/6)<br>
 
-
LaneB: J2-tat-psp-DT 3C5 (9/13)<br>
 
-
LaneC: J2-tat-psp-DT 1C5 (9/15)<br>
 
-
LaneD: J2-tat-psp-DT (9/15)<br>
 
-
LaneE: J2-tat-psp-DT 1C3 (9/18)<br>
 
-
LaneF: J2-tat-psp-DT 3C5 (9/18)<br>
 
-
LaneG: J2-tat-psp-DT-LacP-torA-GFP-DT:2 (9/20)<br>
 
-
LaneH: J2-tat-psp-DT-LacP-torA-GFP-DT:3 (9/20)<br>
 
-
LaneA~H are (E,P)<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!J2-tat-psp-DT-LacPtorA-GFP-DT:2||BufferH||Pst||MilliQ||total
 
-
|-
 
-
|15||3||1||12||30
 
-
|}
 
-
{|class="wikitable"
 
-
!J2-tat-psp-DT-LacP-torAGFP-DT:3||BufferH||Pst||MilliQ||total
 
-
|-
 
-
|15||3||1||12||30
 
-
|}<br>
 
-
 
-
====Restriction enzyme processing====
 
-
 
-
{|class="wikitable"
 
-
!LacP-torAGFP-DT(37.4μg/mL)||BufferH||EcoRI||Pst||total
 
-
|-
 
-
|25||3||1||1||30
 
-
|}<br>
 
-
====Electrophoresis====
 
-
1: J2-tat-psp-DT-LacP-torA-GFP-DT:2<br>
 
-
2: LacP-torA-GFP-DT (E,P) (9/22)<br>
 
-
3: J2-tatpsp-DT-LacP-torA-GFP-DT:3<br>
 
-
 
-
 
-
 
-
 
-
 
-
==September 23==
 
-
====Electrophoresis====
 
-
写真お願いします。
 
-
*LacP-torA-GFP-DT(EcoR1, Pst1)
 
-
lane_1 1kb ladder
 
-
lane_3 DNA
 
-
lane_4 DNA
 
-
lane_5 DNA
 
-
====Gel extraction====
 
-
写真お願いします。
 
-
*J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
 
-
*LacP-torA-GFP-DT(Xba1, Pst1)
 
-
lane_1 1kb ladder
 
-
lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
 
-
lane_5 LacP-torA-GFP-DT(Xba1, Pst1)
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT (78.4μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|15||1||1||3||3||7||30
 
-
|}
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT (78.4μg/mL)||Xba1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|3||0.5||1||1||4.5||10
 
-
|}
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
 
-
|-
 
-
|9||1||1||2||7||20
 
-
|}
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)||pSB4K5 (9.6μg/mL)||Ligation High Ver.2||total
 
-
|-
 
-
|8||4||6||18
 
-
|}
 
-
at 16℃ for 1 hour<br>
 
-
====Liquid culture====
 
-
*LacP-tatABCD-pspA-DT (2, 3, 5, 6)
 
-
====Colony PCR====
 
-
LacP-tatABCD-pspA-DT (1-6)
 
-
{|class="wikitable"
 
-
!2×Quick Taq||VF2||VR||MilliQ||Total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
94℃, 2min<br>
 
-
94℃, 30sec<br>
 
-
55℃, 30sec<br>
 
-
68℃, 4min<br>
 
-
→30cycles<br><br>
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
①, LacP-torA-GFP-DT<br>
 
-
②, LacP-torA-GFP-DT(Xba1, Pst1)<br>
 
-
③, LacP-torA-GFP-DT(Xba1)<br>
 
-
④, LacP-torA-GFP-DT(Pst1)<br>
 
-
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)<br>
 
-
====Liquid culture====
 
-
*LacP-torA-GFP-DT (4, 7)
 
-
*LacP-GFP generater (1-3)
 
-
*T7-6His-GFP-DT (1, 2)
 
-
====Gel extraction====
 
-
写真お願いします。
 
-
*LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)
 
-
====Purification====
 
-
*J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)||LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)||Ligation High Ver.2||total
 
-
|-
 
-
|10||4||7||21
 
-
|}
 
-
{| class="wikitable"
 
-
!J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)||LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)||Ligation High Ver.2||total
 
-
|-
 
-
|8||2||5||15
 
-
|}
 
-
at 16℃ for 1 hour<br>
 
-
====Transformation====
 
-
LacP-torA-GFP-DT(pSB4K5)<br>
 
-
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
 
-
J23107-tatABCD-pspA-DT-LacP-kil-DT<br>
 
-
{| class="wikitable"
 
-
!DNA||competent cell||total
 
-
|-
 
-
|2||10||12
 
-
|}
 
-
preculture at 37℃ for 1 hour
 
-
culture at 37℃ for 16 hours
 
-
====Electrophoresis====
 
-
写真お願いします。<br>
 
-
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 
-
lane_1 1kb ladder
 
-
lane_2~7 DNA
 
-
lane_8 1kb ladder
 
-
 
-
==September 24==
 
-
 
-
 
-
 
-
{{Kyoto/footer}}
 

Revision as of 19:44, 24 September 2012

Contents

September 18

Gel extraction

写真お願いします。

  • LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)

Ligation

J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mLLacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2total
81.59.519
LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2total
1.51.53

at 4℃ for 1 hour

Transformation

  • J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
  • LacP-torA-GFP-DT(EcoR1, Xba1)

Observation through a confocal microscope

We made sample for observation through a confocal microscope We incubated sample with 0mM/0.5mM IPTG for 1.5 hours. Then, we incubated onto medium without IPTG for 2.5 hours.

IPTG(mM)medium
0LB
0.1LB
0.1LB with 0.1mM IPTG
0LB with 2 percent glucose
0.1LB with 2 percent glucose
0LB with 5 percent glucose
0.1LB with 5 percent glucose
0LB with 2 percent glucose
0LB ここ途中
0LB
0LB

Then, we incubated onto medium without IPTG for 2.5 hours.




Restriction enzyme processing

J23101-Kil-DTBufferMXbaIPstBSAMilliQtotal
153113730
J23101-Kil-DTBufferMXbaIBSAMilliQtotal
310.514.510
J23101-Kil-DTBufferHPstMilliQtotal
310.55.510

37℃

Retrieved from "http://2012.igem.org/Team:Kyoto/Secretion/Notebook"