Team:HokkaidoU Japan/Notebook/aggregation Week 2

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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

digestion result


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

digestion result


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.


pT7 + RBS on pSB1K3(Total 2247bp)

Electrophoresis resulsts


Ag43 + dT on pSB1AK3(Total 6444bp)

Electrophoresis resulsts

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 products were reacted in 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

Liquid culturing

Liquid culture for some colonies used in colony PCR.

  1. Prepared 200ul LB solutions.
  2. To these LB solutions, added 6ul of LB solutions (colony PCR solutions were pre-cultivated in about 3hrs) and added 2ml LB and antibiotic(Cp).
  3. Cultivated 18hrs30min.


July 13th

Mini-prep

Mini-prep for colony No.1~8 We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics). Mini-prep result HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg

Digestion

Digestion to confirm how DNA fragments ligated. Digestion Recipe

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


Digestion result

HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg


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


Degree Minute
16 30
65 10
4 Hold

Ligation result with colony no.1 (see colony pcr result in 9th)

HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg

Transformation

Transformation for ligation products written above.

  1. Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
  2. Stood on ice in 30min.
  3. Added 600ul of LB to transformed DH5α solution.
  4. Pre-cultivate in 2hrs
  5. Splead 300ul of LB&DH5α solution to LBC and LBT , 100ul added into 900ul of LB.
  6. Splead 300ul of LB&DH5α solution from 1000ul LB (100ul added into 900ul) to LBC and LBT.
  7. Cultivated in 21hrs.
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 HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg

Liquid culture

Liquid culture for Ag43(BBa_K346007)

  1. Picked up one colony from single colony isolated plate.
  2. Dipped into LBC.
  3. cultivated.


July 15th

Gel extraction

We used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to purify digestion products (see 14th).

Digestion

Digestion to confirm what kind of restriction enzyme cutting sites these DNA (colony 1 and 6) have. EcoRI

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


XbaI

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


PstI

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


SpeI

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


EcoRI + PstI

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


XbaI + SpeI

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

Ethanol precipitation

Ethanol precipitation for digestion products.

  1. Added 2ul of NaoAc, 1.5ul of glycogen and 50ul of 100% ethanol.
  2. Centrifuged in 15000rpm, 10min at 4C.
  3. Remove supernatant and added 220ul of 70% ethanol.
  4. Centrifuged in 15000rpm, 5min at 4C.
  5. Remove supernatant and air drying in room temperature then added 5ul of DW.

Electrophoresis

Electrophoresis for digest-ethanol precipitation products.

  1. added 5ul of EtBr.
  2. Migrated in 30min.
Digestion result Digestion results for pT7-RBS-RFP-dT on pSB1C3 (once digested with EcoRI and PstI) HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg

Single colony isolation

Single colony isolation for Transformation products synthesized yesterday.

  1. one colony picked up from cultivated LBC and LBT plate.
  2. Spread on LBC and LBT.
  3. Cultivated.

Digestion

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

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


dT

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


pT7-RBS

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