Team:Kyoto/Notebook

From 2012.igem.org

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(→Florigen)
(→Secretion)
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{{Kyoto/header}}
{{Kyoto/header}}
=Secretion=
=Secretion=
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<div class="_kyoto-note">
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[[Team:Kyoto/Secretion/Notebook]]
-
==February 7==
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'''Preculture'''  <small>by_???</small><br>
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We started preculture at 12:10.<br>
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-
 
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==February 8==
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-
 
+
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==March 1==
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==March 2==
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==March 3==
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==March 4==
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==March 5==
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==March 6==
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'''Restriction'''<br>
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{| class="wikitable"
+
-
!GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
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-
|12||0.5||0.5||3||0.5||13.5||30
+
-
|-
+
-
|}
+
-
 
+
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==March 7==
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'''Electrophoresis'''[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
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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>
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-
*The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
+
-
'''Ligation'''<br>
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-
{| class="wikitable"
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!torA||pSB1C3||Ligation High Ver.2||total
+
-
|-
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|3||3||3||9
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-
|}
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{| class="wikitable"
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!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
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|4||2||3||9
+
-
|}
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at 4℃, for overnight<br>
+
-
*torA→31.8ng/µL×3µL=95.4ng=0.529pmol<br>
+
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*pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol<br>
+
-
*pspA→49.3ng/µL×4µL=197.2ng=0.308pmol<br>
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*pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol<br>
+
-
 
+
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==March 8==
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'''Restriction'''<br>
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{| class="wikitable"
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!pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
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|20||0.2||0.2||3||0.2||6.4||30
+
-
|-
+
-
|}
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at 37℃ for 1 hour.<br>
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→Purification : 36.6ng/µL<br><br>
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-
'''Ligation'''<br>
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{| class="wikitable"
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-
!Kil||pSB4K5||Ligation High Ver.2||total
+
-
|-
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|10||1||5||16
+
-
|}
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at 4℃ for overnight<br>
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*Kil→37.7ng/µL×10µL=377ng=879fmol<br>
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-
*pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
+
-
'''Liquid culture'''<br>
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Lacp + pSB3C5 -1, 2<br><br>
+
-
'''Transformation'''<br>
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-
{| class="wikitable"
+
-
!torA||pspA||competent cell||total
+
-
|-
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|1||0||10||11
+
-
|-
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-
|0||1||10||11
+
-
|}
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We use commercially available competent cells in this time.<br>
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-
 
+
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==March 9==
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'''Restriction'''<br>
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{| class="wikitable"
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!tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
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|10||0.2||0.2||3||0.3||16.3||30
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-
|}
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at 37℃ for 1hour<br>
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→purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
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-
'''Miniprep'''<br>
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-
lacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
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-
lacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
+
-
'''Colony PCR'''<br>
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-
{| class="wikitable"
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-
!Quick Taq||VF||VR||MilliQ||total
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-
|-
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|25||1||1||23||50
+
-
|}
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-
94℃ 2min<br>
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94℃ 30sec<br>
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55℃ 30sec<br>
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68℃ 6sec<br>
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25cycles<br><br>
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-
'''Ligation'''<br>
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{| class="wikitable"
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-
!tatABCD||constP J23107||Ligation High Ver.2||total
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-
|-
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|5||1||3||9
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-
|}
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tatABCD : 227fmol<br>
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constP J23107 : 21fmol<br><br>
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-
'''Transformation'''
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{| class="wikitable"
+
-
! ||pspA||torA||Kil||competent cell
+
-
|-
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|1||1||0||0||10
+
-
|-
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|2||0||1||0||10
+
-
|-
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|3||0||0||1||10
+
-
|}
+
-
'''Miniprep'''<br>
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4mL of plusgrow which had been cultured for overnight.<br>
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pSB4K5 : 80.5μg/mL<br>
+
-
 
+
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==March 10==
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'''Screening PCR'''<br>
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{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
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|25||1||1||23||50
+
-
|}
+
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[[File:Electrophoresis0310.JPG|400px|thumb|right]]
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-
Then we did electrophoresis to confirm.<br>
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1.1kb ladder<br>
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2.kil (649bp)<br>
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3,4,5, pspA (969bp)<br>
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6,1kb ladder<br><br>
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1, 100bp ladder<br>
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2,3,4, torA signal<br><br>
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'''Restriction'''<br>
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-
{| class="wikitable"
+
-
!LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
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|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
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for 2.5 hours at 37℃<br>
+
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→purification  29.0ng/μL<br>
+
-
{| class="wikitable"
+
-
!torA||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
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|10||0.2||0.2||2||0.2||7.4||20
+
-
|}
+
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for 2.5 hours at 37℃<br>
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→purification  91.8ng/μL<br><br>
+
-
'''Ligation'''<br>
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{| class="wikitable"
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-
!torA||Lacp-pSB3C5||Ligation High Ver.2||total
+
-
|-
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|4||2||3||9
+
-
|}
+
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torA : 929fmol<br>
+
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Lacp-pSB3C5 : 284fmol<br>
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for overnight at 4℃
+
-
 
+
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==March 11==
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'''Miniprep'''<br>
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We used 3μL of plus grow that we had cultured for overnight.<br>
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torA : 62.8ng/μL<br><br>
+
-
'''Screening PCR'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VF||VR||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
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'''Electrophoresis'''<br>
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[[File:Electrophoresis031101.JPG|400px|thumb|right]]
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1. 1kb ladder<br>
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2〜11. pspA (969bp)<br>
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12. 1kb ladder<br>
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<br>The results were shown as photograph in the right.<br>
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<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>
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[[File:Electrophoresis031102.JPG|400px|thumb|right]]<br>
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1.1kb ladder<br>
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2. pspA (PCR product)<br>
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3, pspA (Eco, Spe)<br>
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4〜6, pspA (colony PCR)<br>
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7,1kb ladder<br>
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<br>The results were shown as photograph in the right.<br>
+
-
 
+
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==March 12==
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'''Transformation'''<br>
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-
{| class="wikitable"
+
-
!DT(1ng/μL)||DT(0.1ng/μL)||Kil||lacP-torA||MilliQ||competent cell||total
+
-
|-
+
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|1||0||0||0||0||20||21
+
-
|-
+
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|0||1||0||0||0||20||21
+
-
|-
+
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|0||0||5||0||0||50||51
+
-
|-
+
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|0||0||0||5||0||50||51
+
-
|-
+
-
|0||0||0||0||1||20||21
+
-
|}
+
-
'''Restriction'''<br>
+
-
{| 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'''<br>
+
-
{| class="wikitable"
+
-
! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||total
+
-
|-
+
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|1|4||2||0||3||9
+
-
|-
+
-
|2|2||2||0||2||6
+
-
|-
+
-
|3|0||2||2||2||6
+
-
|}
+
-
 
+
-
==March 13==
+
-
'''Miniprep'''
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-
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'''<br>
+
-
{| 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'''<br>
+
-
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'''<br>
+
-
We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
+
-
→6,8,9,10 were succeeded.<br><br>
+
-
'''Restriction'''<br>
+
-
{| 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'''<br>
+
-
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'''<br>
+
-
Kil, pspA and torA<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||Primer-r||Primer-f||MilliQ||total
+
-
|-
+
-
|25||1||1||23||50
+
-
|}
+
-
 
+
-
==March 16==
+
-
'''Miniprep'''<br>
+
-
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'''<br>
+
-
{| 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'''<br>
+
-
[[File:Electrophoresis0316.JPG|400px|thumb|right]]
+
-
The results were shown as photograph in the right.<br><br>
+
-
'''Checking Transformation Efficiency'''<br>
+
-
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'''<br>
+
-
{| 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'''<br>
+
-
{| 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'''<br>
+
-
{| 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'''<br>
+
-
{| class="wikitable"
+
-
!Kil||DT||Ligation High Ver.2||total
+
-
|-
+
-
|10||2||6||18
+
-
|}
+
-
We did this for an hour at 16℃.<br><br>
+
-
'''Restriction'''<br>
+
-
{| 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'''<br>
+
-
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
+
-
 
+
-
==March 20==
+
-
'''Screaning PCR'''<br>
+
-
{| 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'''<br>
+
-
{| 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'''<br>
+
-
GFP-DT-1 : 77.7ng/µL<br>
+
-
GFP-DT-2 : 67.6ng/µL<br><br>
+
-
'''Restriction'''<br>
+
-
{| 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'''<br>
+
-
{| 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'''<br>
+
-
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'''<br>
+
-
{| 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'''<br>
+
-
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'''<br>
+
-
{| 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'''<br>
+
-
pspA 11.5ng/µL.<br><br>
+
-
'''Miniprep'''<br>
+
-
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'''<br>
+
-
{| 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'''<br>
+
-
Lacp+pSB3C5-8  40.4µg/mL<br>
+
-
Kil+DT-4  26.8µg/mL<br>
+
-
 
+
-
==March 26==
+
-
'''Miniprep'''<br>
+
-
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'''<br>
+
-
{| class="wikitable"
+
-
!Lacp-Kil-DT||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
 
+
-
==March 27==
+
-
'''Miniprep'''    <small>by </small>
+
-
We retryed miniprep of torA(pSB1C3).<br>
+
-
We got torA and its concentration was 39.3[ng/µL].<br><br>
+
-
'''Transformation'''<br>
+
-
{| 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'''<br>
+
-
{| 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'''<br>
+
-
Lacp-torA-GFP-DT<br>
+
-
</div>
+
=Florigen=
=Florigen=

Revision as of 05:22, 7 September 2012

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Contents

Secretion

Team:Kyoto/Secretion/Notebook

Florigen

Team:Kyoto/Florigen/Notebook

Golden Gate Assembly

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

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