Team:HokkaidoU Japan/Notebook/Week 2

From 2012.igem.org

(Difference between revisions)
(July 11th)
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#50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.  
#50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.  
#Cultivated.
#Cultivated.
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<br />
 
</div>
</div>
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==July 12th==
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<div class="hokkaidou-notebook-daily">
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 +
;Colony PCR
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Colony PCR for ligation product.Each product reacted recipes written below.
 +
#picked up each 16 colonies from LB plates by Autoclaved toothpicks.
 +
#Dipped into 10ul DW in 1.5ml eppendorf tubes.
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#from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
 +
#Ran PCR machine in recipe below.
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#Electrophoresis for confirmation of PCR results.
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 +
 +
PCR reaction solution
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{|class="hokkaidou-table-pcr-reagent"
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|-
 +
|DNA solution
 +
|4ul
 +
|-
 +
|KapaTaq ready mix
 +
|5ul
 +
|-
 +
|BioBrick prefix forward primer
 +
|0.5ul
 +
|-
 +
|BioBrick suffix reverse primer
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|0.5ul
 +
|-
 +
|Total
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|10ul
 +
|}
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 +
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'''PCR recipe'''
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 +
(pT7 + RBS)
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{|class="hokkaidou-table-pcr-time"
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|-
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|Number
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|Degree
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|Second
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|-
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|1
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|94
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|120
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|-
 +
|2
 +
|94
 +
|30
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|-
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|3
 +
|68
 +
|90
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|-
 +
|4
 +
|4
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|HOLD
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|}
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Cycle:2~3 x 40

Revision as of 10:39, 12 July 2012

Contents

July 9th

pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) ligation products were transformed and cultivated then some colonies were formed (12~16 colonies) so we confirmed really insert DNA (3A:pT7 and RBS, standard:Ag43 and dT) were inserted to vector or not by colony PCR.

Colony PCR

Colony PCR for assembly products.Each product reacted recipes written below.

  1. picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
  2. Dipped into 10ul DW in 1.5ml eppendorf tubes.
  3. from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
  4. Ran PCR machine in recipe below.
  5. Electrophoresis for confirmation of PCR results.


PCR reaction solution

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


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. Extension step needed >180seconds.

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

Cycle:2~3 x 35


Electrophoresis results

Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 1% agarose gel.


pT7 + RBS on pSB1K3 bbp-Insert-bbs:86bp

HokkaidoU2012 120709 pt7-rbs 75% scale.jpg


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

HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg


We couldn't confirm insert DNA were really ligated with Vector or not. Next we tried confirmation of insert DNA by Electrophoresis of mini-prep products. For mini-prep, we needed do liquid culture.


Liquid culturing

Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).

  1. Prepared 1800ul LB solutions.
  2. To these LB solutions, added 200ul of LB solutions (colony PCR solutions were pre-cultivated in about 2hrs) and each antibiotics(Km (for (pT7 + RBS) on pSB1K3) and Amp(for (Ag43 + dT) on pSB1AK3)).
  3. Cultivated 15hrs30min.


July 10th

Mini-prep

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 15hrs 30min.

  1. Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
  2. Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.

Electrophoresis resulsts

pT7 + RBS on pSB1K3(Total 2247bp)

HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg


Ag43 + dT on pSB1AK3(Total 6444bp)

HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg

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

Digestion

Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P.


Digestion recipe pT7-RBS

pT7-RBS 1,5ul
EcoRI 1ul
PstI 1ul
10xH buffer 2ul
DW 14.5ul
Total 20ul


Digestion recipe Ag43-dT

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


Digestioned at 37c in 2hrs.


Digestion results

pT7+RBS

HokkaidoU2012 120710 pt7-rbs,digestion.jpg


Ag43+dT

HokkaidoU2012 120710 ag43-dt,digestion.jpg


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


Ag43-dT

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


pSB1C3

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

Reacted in 2hrs at 37c.

July 11th

Ligation

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


Ligation recipe

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


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold


Electrophoresis result

HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg


Transformation

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

  1. Added DNA soltions (Ligation products) 1ul to BL21(DE3) compitent cell.
  2. Stood on ice in 30min.
  3. Heatshock for 1min at 42c.
  4. Added 200ul of LB to transformed BL21(DE3) solution.
  5. Pre-cultivate in 2hrs
  6. Splead 200ul of LB&BL21(DE3) solution supernant to LBC.
  7. 50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.
  8. Cultivated.

July 12th

Colony PCR

Colony PCR for ligation product.Each product reacted recipes written below.

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


PCR reaction solution

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


PCR recipe

(pT7 + RBS)

Number Degree Second
1 94 120
2 94 30
3 68 90
4 4 HOLD
Cycle:2~3 x 40