Team:HokkaidoU Japan/Notebook/aggregation Week 2

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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==July 9th==
+
===July 9th===
-
<div>
+
<div class="hokkaidou-section">
-
pT7 + RBS (3A Assembly) and  Ag43 + dT (standard assembly) ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.
+
pT7 + RBS (3A Assembly) and  Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.
-
===Colony PCR===
+
====Colony PCR====
-
<p>
+
Colony PCR for assembly products.
Colony PCR for assembly products.
#Picked up colony from LB plates by Autoclaved toothpicks.
#Picked up colony from LB plates by Autoclaved toothpicks.
-
#re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.
+
#Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.
#4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.
#Ran PCR machine in recipe below.
#Ran PCR machine in recipe below.
-
 
PCR reaction solution
PCR reaction solution
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|}
|}
Cycle:2~3 x 35
Cycle:2~3 x 35
-
</p>
 
-
===Electrophoresis results===
+
 
-
<p>
+
====Electrophoresis results====
 +
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]
 +
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br />
Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.<br />
-
 
-
 
pT7 + RBS on pSB1K3
pT7 + RBS on pSB1K3
bbp-Insert-bbs:86bp
bbp-Insert-bbs:86bp
-
 
-
[[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|PCR result]]
 
-
 
Ag43 + dT on pSB1AK3
Ag43 + dT on pSB1AK3
bbp-Insert-bbs:3290bp
bbp-Insert-bbs:3290bp
-
 
-
[[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|PCR result]]
 
We couldn't confirm insert DNA were really ligated with Vector or not.
We couldn't confirm insert DNA were really ligated with Vector or not.
-
Next step, we tried confirmation of insert DNA by Electrophoresis of mini-prep products.  
+
Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids.  
-
For mini-prep, we needed do liquid culture.
+
For plasmid extraction, we needed to incubate in liquid culture.
-
</p>
+
-
===Incubate for mini-prep===
+
====Incubate for plasmid extraction====
-
<p>
+
#Prepared 1800 ul LB solutions.
#Prepared 1800 ul LB solutions.
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).
#Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).
#Incubated for 15 hrs and 30 min.
#Incubated for 15 hrs and 30 min.
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
 
+
===July 10th===
-
==July 10th==
+
<div class="hokkaidou-section">
-
<div>
+
====Plasmid extraction====
-
===Mini-prep===
+
Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.
-
<p>
+
#Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).
-
Mini-prep for some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)cultivated in LBA(Ag43 + dT) and LBK(pT7 + RBS) in 15 hrs and 30 min.
+
-
#Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
+
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.
#Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.
-
</p>
 
-
 
pT7 + RBS on pSB1K3(Total 2247bp)
pT7 + RBS on pSB1K3(Total 2247bp)
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]
[[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]
-
 
-
 
Ag43 + dT on pSB1AK3(Total 6444bp)
Ag43 + dT on pSB1AK3(Total 6444bp)
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]
[[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]
-
To confirm more about insert DNA, we tried to digest these DNA with EcoRI and PstI.
+
To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.
-
===Digestion===
+
====Digestion====
-
<p>
+
Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.<br />
-
Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P.
+
Digestion recipe<br />
-
Digestion recipe
+
pT7-RBS
pT7-RBS
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
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-
Digestion recipe
+
Digestion recipe<br />
Ag43-dT
Ag43-dT
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 193: Line 174:
-
Digestioned at 37c in 2 hrs.
+
Digestioned at 37c for 2 hrs.
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]
[[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg|thumb|pT7-RBS digestion results]]
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]
[[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg|thumb|Ag43-dT digestion results]]
 +
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]
-
Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]
+
====Digestion====
-
</p>
+
Digestion for 3A Assembly.<br />
-
 
+
-
===Digestion===
+
-
<p>
+
-
Digestion for 3A Assembly.
+
-
 
+
-
 
+
pT7-RBS
pT7-RBS
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
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|}  
|}  
-
Reacted in 2 hrs at 37c.
+
Reacted for 2 hrs at 37c.
-
</p>
+
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 11th==
+
===July 11th===
-
<div>
+
<div class="hokkaidou-section">
-
===Ligation===
+
====Ligation====
-
<p>
+
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)<br />
-
Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)
+
-
 
+
-
 
+
Ligation recipe
Ligation recipe
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
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|}
|}
-
</p>
+
 
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]
[[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg|thumb|ligation result]]
-
===Transformation===
+
====Transformation====
-
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coadin site in their genomic DNA).
+
Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).
-
#Added DNA soltions (Ligation products) 1 ul to BL21(DE3) compitent cell.
+
#Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).
-
#Stood on ice in 30 min.
+
#Incubated on ice for 30 min.
-
#Heatshock for 1min at 42c.
+
#Heatshock for 1 min at 42c.
#Added 200 ul of LB to transformed BL21(DE3) solution.
#Added 200 ul of LB to transformed BL21(DE3) solution.
-
#Pre-cultivate in 2 hrs
+
#Pre-incubate for 2 hrs
-
#Splead 200 ul of LB&BL21(DE3) solution supernant to LBC.
+
#Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.
-
#50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200 ul to LBC plate.  
+
#50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate.  
-
#Cultivated.
+
#Incubated for 23 hrs and 30 minutes.
-
</p>
+
<br style="line-height: 0; clear: both;" />
-
</div></div>
+
 +
</div></div>
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 12th==
+
===July 12th===
-
<div>
+
<div class="hokkaidou-section">
-
===Colony PCR===
+
====Colony PCR====
-
<p>
+
Colony PCR for ligation product.Each products were reacted in recipes written below.  
Colony PCR for ligation product.Each products were reacted in recipes written below.  
-
#picked up each 16 colonies from LB plates by Autoclaved toothpicks.
+
#Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.
#Dipped into 10 ul DW in 1.5 ml eppendorf tubes.
-
#from 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.
+
#From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.
#Ran PCR machine in recipe below.
#Ran PCR machine in recipe below.
#Electrophoresis for confirmation of PCR results.
#Electrophoresis for confirmation of PCR results.
Line 402: Line 373:
|}
|}
Cycle:2~3 x 40
Cycle:2~3 x 40
-
</p>
 
-
===Liquid culturing===
+
 
-
<p>
+
====Liquid culture====
Liquid culture for some colonies used in colony PCR.
Liquid culture for some colonies used in colony PCR.
#Prepared 200 ul LB solutions.
#Prepared 200 ul LB solutions.
-
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-cultivated in about 3 hrs) and added 2 ml LB and antibiotic(Cp).
+
#To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).
-
#Cultivated 18 hrs and 30 min.
+
#Incubated for 18 hrs and 30 min.
-
</p>
+
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 13th==
+
===July 13th===
-
<div>
+
<div class="hokkaidou-section">
-
===Mini-prep===
+
====Plasmid extraction====
-
<p>
+
Plasmid extraction from colony No. 1~8.<br />
-
Mini-prep for colony No. 1~8
+
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.
-
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics).
+
-
 
+
-
 
+
-
Mini-prep result
+
-
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg]]
+
[[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg|thumb|Plasmid extraction results]]
-
</p>
+
-
===Digestion===
+
====Digestion====
-
<p>
+
Digestion to confirm how DNA fragments ligated.
Digestion to confirm how DNA fragments ligated.
-
 
+
<br />
-
 
+
Digestion Recipe
Digestion Recipe
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 455: Line 417:
|}  
|}  
 +
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg|thumb|Digestion results]]
 +
<br style="line-height: 0; clear: both;" />
-
Digestion result
 
-
 
-
[[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg]]
 
-
</p>
 
</div></div>
</div></div>
 +
<div class="hokkaidou-notebook-daily">
-
<div class="hokkaidou-notebook-daily">
+
===July 14th===
-
==July 14th==
+
<div class="hokkaidou-section">
-
<div>
+
====Ligation====
-
===Ligation===
+
-
<p>
+
Ligation for digestion fragments written above.
Ligation for digestion fragments written above.
-
 
+
<br />
-
Ligation recipe
+
Ligation recipe<br />
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.
-
 
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
Line 505: Line 463:
|}
|}
-
Ligation result
+
We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.
-
with colony no. 1 (see colony pcr result in 9th)
+
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg|thumb|ligation product]]
-
[[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg]]
 
-
</p>
 
-
===Transformation===
+
====Transformation====
-
<p>
+
Transformation for ligation products written above.
Transformation for ligation products written above.
-
 
+
#Added DNA solutions (Ligation products) 1 ul to DH5&alpha; compitent cell.
-
 
+
#Incubated on ice for 30 min.
-
#Added DNA soltions (Ligation products) 1 ul to DH5&alpha; compitent cell.
+
-
#Stood on ice in 30 min.
+
#Added 600 ul of LB to transformed DH5&alpha; solution.
#Added 600 ul of LB to transformed DH5&alpha; solution.
-
#Pre-cultivate in 2 hrs
+
#Pre-incubate for 2 hrs
-
#Splead 300 ul of LB&DH5&alpha; solution to LBC and LBT , 100 ul added into 900 ul of LB.
+
#Spread 300 ul of LB&DH5&alpha; solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.
-
#Splead 300 ul of LB&DH5&alpha; solution from 1000 ul LB (100 ul added into 900 ul) to LBC and LBT.
+
#Spread 300 ul of LB&DH5&alpha; solution from 1000 ul LB (made at 5) to LBC and LBT plate.
-
#Cultivated in 21 hrs.  
+
#Incubated for 21 hrs.
 +
And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and  PstI once more time.
 +
Showed the result.
 +
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg|thumb|digestion results]]
-
 
+
====Liquid culture====
-
and to get more conformation about pT7-RBS-Ag43-dT on pSB1C3 was really ligated, we tried digest this DNA with EcoRI and  PstI once more time.
+
-
 
+
-
result
+
-
 
+
-
[[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg]]
+
-
</p>
+
-
 
+
-
===Liquid culture===
+
-
<p>
+
Liquid culture for Ag43(BBa_K346007)
Liquid culture for Ag43(BBa_K346007)
-
#Picked up one colony from single colony isolated plate.
+
#Picked up one colony from plate done single colony isolation.
-
#Dipped into LBC.
+
#Dipped into LBC solution.
-
#cultivated.
+
#Incubated for 16 hrs.
-
</p>
+
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 15th==
 
-
<div>
 
-
===Gel extraction===
 
-
<p>
 
-
We used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to purify digestion products (see 14th).
 
-
</p>
 
-
===Digestion===
+
===July 15th===
-
<p>
+
<div class="hokkaidou-section">
-
Digestion to confirm what kind of restriction enzyme cutting sites these DNA (colony 1 and 6) have.  
+
====Gel extraction====
 +
Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).
 +
====Digestion====
 +
Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6).
 +
<br />
EcoRI
EcoRI
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 683: Line 627:
|20 ul
|20 ul
|}
|}
-
</p>
 
-
===Ethanol precipitation===
+
 
-
<p>
+
====Ethanol precipitation====
Ethanol precipitation for digestion products.
Ethanol precipitation for digestion products.
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
-
#Centrifuged in 15000 rpm, 10 min at 4C.
+
#Centrifuged at 15000 rpm, 10 min at 4C.
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
#Removed supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000 rpm, 5 min at 4C.
+
#Centrifuged at 15000 rpm, 5 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 5 ul of DW.
+
#Removed supernatant and dried out at room temperature, after that added 5 ul of DW.
-
</p>
+
-
===Electrophoresis===
+
====Electrophoresis====
-
<p>
+
Electrophoresis for digest-ethanol precipitation products.
Electrophoresis for digest-ethanol precipitation products.
-
#added 5 ul of EtBr.
+
#Added 5 ul of EtBr.
-
#Migrated in 30 min.
+
#Migrated for 30 min.
 +
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg|thumb|digestion results]]
-
'''Digestion result'''
+
====Single colony isolation====
 +
Single colony isolation for transformation products of yesterday.
 +
#Picked up one colony from incubated LBC and LBT plate.
 +
#Spread it on LBC and LBT plate.
 +
#Incubated for 16 hrs.
-
Digestion results for pT7-RBS-RFP-dT on pSB1C3
+
====Digestion====
-
(once digested with EcoRI and PstI)
+
-
 
+
-
[[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg]]
+
-
</p>
+
-
 
+
-
===Single colony isolation===
+
-
<p>
+
-
Single colony isolation for Transformation products synthesized yesterday.
+
-
#one colony picked up from cultivated LBC and LBT plate.
+
-
#Spread on LBC and LBT.
+
-
#Cultivated.
+
-
</p>
+
-
 
+
-
===Digestion===
+
-
<p>
+
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).
Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).
-
 
+
<br />
Ag43
Ag43
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 787: Line 718:
|20 ul
|20 ul
|}
|}
-
</p>
+
 
</div></div>
</div></div>
 +
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
</div>
</div>
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<br style="line-height: 0; clear: both;" />
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 01:52, 27 September 2012

Contents

July 9th

pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) of ligation products were transformed and incubated. We confirmed ideal insert DNA (pT7 and RBS or Ag43 and dT) were inserted by colony PCR.

Colony PCR

Colony PCR for assembly products.

  1. Picked up colony from LB plates by Autoclaved toothpicks.
  2. Re-suspension into 10 ul DW in 1.5 ml eppendorf tubes.
  3. 4 ul was added in PCR reaction solution, and 6 ul was mixed with 200 ul LB.
  4. Ran PCR machine in recipe below.

PCR reaction solution

DNA solution 4 ul
KapaTaq ready mix 5 ul
BioBrick prefix forward primer 0.5 ul
BioBrick suffix reverse primer 0.5 ul
Total 10 ul


PCR recipe (pT7 + RBS)

Number Degree Second
1 94 120
2 94 30
3 68 60
4 4 HOLD

Cycle:2~3 x 40


(Ag43 + dT) Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp.

Number Degree Second
1 94 120
2 94 30
3 68 180
4 4 HOLD

Cycle:2~3 x 35


Electrophoresis results

PCR result
PCR result

Electrophoresis for (pT7 + RBS) by 2% agarose gel and (Ag43 + dT) by 1% agarose gel.
pT7 + RBS on pSB1K3 bbp-Insert-bbs:86bp

Ag43 + dT on pSB1AK3 bbp-Insert-bbs:3290bp


We couldn't confirm insert DNA were really ligated with Vector or not. Next step, we tried confirmation of insert DNA by electrophoresis of extracted plasmids. For plasmid extraction, we needed to incubate in liquid culture.

Incubate for plasmid extraction

  1. Prepared 1800 ul LB solutions.
  2. Mixed 200 ul of cultures (suspention were incubated for 2 hrs) and antibiotics(Km (pT7-rbs on pSB1K3), Amp(Ag43-dT on pSB1AK3)).
  3. Incubated for 15 hrs and 30 min.

July 10th

Plasmid extraction

Extracted plasmids from some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No. 4, 5, 9, 10 colonies and Ag43 + dT were No. 1, 2, 3, 4 colonies)incubated in LBA(Ag43 + dT) and LBK(pT7 + RBS) for 15 hrs and 30 min.

  1. Extracted from LB culturing products by using FastGene Plasmid Mini kit(Nippon Genetics).
  2. Electrophoresis (Used pre-migrated 1% agarose gels with 5 ul EtBr)in 30 min.

pT7 + RBS on pSB1K3(Total 2247bp)

Electrophoresis resulsts

Ag43 + dT on pSB1AK3(Total 6444bp)

Electrophoresis resulsts

To confirm more correctly about insert DNA, we tried to digest these DNA with EcoRI and PstI.

Digestion

Digestion for confirmation of insert DNA. Each exracted DNA were digested with E & P.
Digestion recipe
pT7-RBS

pT7-RBS 1.5 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 14.5 ul
Total 20 ul


Digestion recipe
Ag43-dT

Ag43-dT 4 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul


Digestioned at 37c for 2 hrs.

pT7-RBS digestion results
Ag43-dT digestion results

Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS+Ag43-dT+pSB1C3]

Digestion

Digestion for 3A Assembly.
pT7-RBS

DNA 17 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 3 ul
DW 8 ul
Total 30 ul


Ag43-dT

DNA 12.5 ul
XbaI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 3.5 ul
Total 20 ul


pSB1C3

DNA 20 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 3 ul
DW 5 ul
Total 30 ul

Reacted for 2 hrs at 37c.

July 11th

Ligation

Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)
Ligation recipe

pT7-RBS 2 ul
Ag43-dT 2 ul
pSB1C3 3 ul
Ligation Mighty Mix(TAKARA) 8 ul
Total 16 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold


ligation result

Transformation

Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coading site in their genome).

  1. Added DNA solutions (Ligation products) 1 ul to compitent cell of BL21(DE3).
  2. Incubated on ice for 30 min.
  3. Heatshock for 1 min at 42c.
  4. Added 200 ul of LB to transformed BL21(DE3) solution.
  5. Pre-incubate for 2 hrs
  6. Spread 200 ul of supernant of LB&BL21(DE3) solution to LBC plate.
  7. 50ul of LB&BL21(DE3) solution was added to 200ul LB solution then spread 200 ul to LBC plate.
  8. Incubated for 23 hrs and 30 minutes.


July 12th

Colony PCR

Colony PCR for ligation product.Each products were reacted in recipes written below.

  1. Picked up each 16 colonies from LBC plates by Autoclaved toothpicks.
  2. Dipped into 10 ul DW in 1.5 ml eppendorf tubes.
  3. From 10 ul DW, 4 ul was added in PCR reaction solution (below), 6 ul was mixed with 200 ul LB.
  4. Ran PCR machine in recipe below.
  5. Electrophoresis for confirmation of PCR results.


PCR reaction solution

DNA solution 4 ul
KapaTaq ready mix 5 ul
BioBrick prefix forward primer 0.5 ul
BioBrick suffix reverse primer 0.5 ul
Total 10 ul


PCR recipe

(pT7 + RBS)

Number Degree Second
1 94 120
2 94 30
3 68 90
4 4 HOLD

Cycle:2~3 x 40


Liquid culture

Liquid culture for some colonies used in colony PCR.

  1. Prepared 200 ul LB solutions.
  2. To these LB solutions, added 6 ul of LB solutions (colony PCR solutions were pre-incubated for 3 hrs) and added 2 ml LB and antibiotic(Cp).
  3. Incubated for 18 hrs and 30 min.

July 13th

Plasmid extraction

Plasmid extraction from colony No. 1~8.
We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics), and got 50 ul of DNA solution.

Plasmid extraction results

Digestion

Digestion to confirm how DNA fragments ligated.
Digestion Recipe

DNA 4 ul
EcoRI 0.5 ul
PstI 0.5 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul
Digestion results


July 14th

Ligation

Ligation for digestion fragments written above.
Ligation recipe
Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.

Insert 5 ul
Vector 1 ul
Ligation Mighty Mix(TAKARA) 6 ul
Total 12 ul


Degree Minute
16 30
65 10
4 Hold

We tried electrophoresis of colony no. 1 (see colony pcr result at 9th) from ligation product.

ligation product


Transformation

Transformation for ligation products written above.

  1. Added DNA solutions (Ligation products) 1 ul to DH5α compitent cell.
  2. Incubated on ice for 30 min.
  3. Added 600 ul of LB to transformed DH5α solution.
  4. Pre-incubate for 2 hrs
  5. Spread 300 ul of LB&DH5α solution to LBC and LBT plate, and 100 ul was added into 900 ul of LB.
  6. Spread 300 ul of LB&DH5α solution from 1000 ul LB (made at 5) to LBC and LBT plate.
  7. Incubated for 21 hrs.

And to confirm success of ligation about pT7-RBS-Ag43-dT on pSB1C3, we tried to digest this DNA with EcoRI and PstI once more time. Showed the result.

digestion results

Liquid culture

Liquid culture for Ag43(BBa_K346007)

  1. Picked up one colony from plate done single colony isolation.
  2. Dipped into LBC solution.
  3. Incubated for 16 hrs.


July 15th

Gel extraction

Used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to extract digestion products (see July 14th).

Digestion

Digestion to confirm that what kind of restriction enzyme cutting sites are there in DNA (colony 1 and 6).
EcoRI

DNA solution 6 ul
EcoRI 1 ul
10xH buffer 1 ul
DW 2 ul
Total 10 ul


XbaI

DNA solution 6 ul
XbaI 1 ul
10xM buffer 1 ul
BSA 1 ul
DW 1 ul
Total 10 ul


PstI

DNA solution 6 ul
PstI 1 ul
10xH buffer 1 ul
DW 2 ul
Total 10 ul


SpeI

DNA solution 6 ul
SpeI 1 ul
10xM buffer 1 ul
DW 2 ul
Total 10 ul


EcoRI + PstI

DNA solution 6 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 1 ul
DW 11 ul
Total 20 ul


XbaI + SpeI

DNA solution 6 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 1 ul
DW 11 ul
Total 20 ul


Ethanol precipitation

Ethanol precipitation for digestion products.

  1. Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 10 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 5 min at 4C.
  5. Removed supernatant and dried out at room temperature, after that added 5 ul of DW.

Electrophoresis

Electrophoresis for digest-ethanol precipitation products.

  1. Added 5 ul of EtBr.
  2. Migrated for 30 min.
digestion results

Single colony isolation

Single colony isolation for transformation products of yesterday.

  1. Picked up one colony from incubated LBC and LBT plate.
  2. Spread it on LBC and LBT plate.
  3. Incubated for 16 hrs.

Digestion

Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).


Ag43

DNA solution 9 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 7 ul
Total 20 ul


dT

DNA solution 1.1 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 14.9 ul
Total 20 ul


pT7-RBS

DNA solution 6 ul
EcoRI 1 ul
10xH buffer 2 ul
DW 11 ul
Total 20 ul