Team:HokkaidoU Japan/Notebook/aggregation Week 7
From 2012.igem.org
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).
- Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30min.
- Added 350 ul of LB.
- Prepared and Labeled two petri dishes with LBA.
- Plate 300 ul of the transformation onto first dish and spread.
- Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
- 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 |
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.
In this result, N2 has about 500bp band, but N1 has not it.
So, we use to liquid culture, No. 7,10,12.
We cultured these DNA in E. coli solution, after added 200 ul LB, for 37C
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours in 37C.
Transformation
Transformation of BBa_I13453 (pBAD on pSB1A3) in E. coli(DH5α).
- Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Added 350 ul of LB.
- Prepared and Labeled two petri dishes with LBK.
- Plate 300 ul of the transformation onto first dish and spread.
- Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
- Incubated the plates at 37C for 19 hours.
Digestion
According to sequencing results, we found that we failed to make pT7-RBS on pSB1K3.
So, we tried to 3A assembry again.
When the restriction enzyme digest the DNA, it is important for 3A assembry to digest completly. So, we did activate the restriction enzymes for 10 hours.
pT7 (BBa_I719005)
EcoRI/SpeI
DNA solution (40 ng/ul) | 12.5 ul |
EcoRI | 1 ul |
SpeI | 1 ul |
10xH buffer | 2 ul |
DW | 3.5 ul |
Total | 20 ul |
RBS (BBa_B0034) XbaI/PstI
DNA solution (40 ng/ul) | 12.5 ul |
XbaI | 1 ul |
PstI | 1 ul |
10xM buffer | 2 ul |
DW | 3.5 ul |
Total | 20 ul |
pSB1C3 EcoRI/PstI
DNA solution (25 ng/ul) | 12 ul |
EcoRI | 1 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 4 ul |
Total | 20 ul |