Team:HokkaidoU Japan/Notebook/aggregation Week 7

From 2012.igem.org

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[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]
[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]
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==Gel extraction==
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Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
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Revision as of 06:17, 17 August 2012

Contents

August 16th

ligation

We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.

pT7-RBS (5 ng/ul) 1 ul
Ag43-dT (25 ng/ul) 2 ul
Ligation Mighty Mix(TAKARA) 5 ul
DW 2 ul
Total 10 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold

Transformation

Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(DH5α).

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30min.
  3. Added 350 ul of LB.
  4. Prepared and Labeled two petri dishes with LBA.
  5. Plate 300 ul of the transformation onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for hours.

Digestion

Ag43-dT on pSB1AK3 digestion with SpeI and XbaI. Ag43-dT SpeI and XbaI

DNA solution (100 ng/ul) 12 ul
SpeI 1 ul
XbaI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul

pBad-RBS on pSB1A3 electrophoresis result
digestion result

Gel extraction

Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.


August 17th

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not.

DNA solution 4 ul (1 colony/10 ul DW)
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer (50 pmol/ul)) 0.5 ul
Reverse Primer(PS-R primer (50 pmol/ul)) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls. Desired product is about 800~1000bp.

[[image:|thumb|Colony PCR result]]

We thought that colonies of No. have band. Next step, we resuspended colonies and cultured (add 1700 ul LB and 2 ul Amp) for hours in 37C.