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Team:DTU-Denmark/Notebook/29 August 2013
From 2013.igem.org
29 August 2013
Contents |
Lab 208
Main purpose
- Biobrick preparation
Who was in the lab
Henrike, Kristian, Julia
Procedure
Introduce endings for USER cloning in pSB1C3 for Nir construct
To make the transportation vector for HAO, AMO TAT construct and so on.
Reactions were made with pSB1C3 template from a PCR purification(Pur) and the batch that we got from iGEM HQ(HQ). 13 samples was made with each template and 6 was run on 58C annealing the other 7 was run on a TouchDown PCR. Sample composition in 58C PCR:
- GC + 2% DMSO
- GC + GC 4% DMSO
- GC 1M betaine
- GC + 5% DMSO
- GC + 4% DMSO + 1uL 50mM MgCl2
- GC + 4% DMSO + 2uL 50mM MgCl2
Samples on the TouchDown PCR:
- GC
- GC + 2% DMSO
- GC + GC 4% DMSO
- GC 1M betaine
- GC + 5% DMSO
- GC + 4% DMSO + 1uL 50mM MgCl2
- GC + 4% DMSO + 2uL 50mM MgCl2
The program for the 58C PCR was like following:
| Temperature | time(min:sec) | cycles |
|---|---|---|
| 98C | 2:00 | 1 |
| 98C | 0:10 | 35 |
| 58C | 1:00 | 35 |
| 72C | 2:00 | 35 |
| 72C | 5:00 | 1 |
| 10C | infinity | 1 |
The TouchDown PCR program was like the following:
| Temperature | time(min:sec) | cycles | temp. increment |
|---|---|---|---|
| 98C | 2:00 | 1 | |
| 98C | 0:10 | 14 | |
| 65C | 1:00 | 14 | -0.5 |
| 72C | 2:00 | 14 | |
| 98C | 0:10 | 25 | |
| 58C | 1:00 | 25 | |
| 72C | 2:00 | 25 | |
| 72C | 5:00 | 1 | |
| 10C | infinity | 1 |
Introduce endings for USER cloning in pSB1C3 for Nir construct
In order to introduce our constructs in the biobrick vector we need to introduce special endings that are necessary for the USER cloning reaction. The table shows the composition of each reaction:
| component | 1 | 2 | 3 | Neg |
|---|---|---|---|---|
| dNTPs | 1uL | 1uL | 1uL | 1uL |
| HF buffer | 10uL | 10uL | 10uL | 10uL |
| X7 polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
| MilliQ water | 28.5uL | 28.5uL | 28.5uL | 29.5uL |
| template pSB1C3 | 1uL | 1uL | 1uL | - |
| FW primer 57a2 | 3uL | 3uL | 3uL | 3uL |
| RV primer 57b | 3uL | 3uL | 3uL | 3uL |
| DMSO (100%) | 2uL (4%) | 2uL (4%) | 2uL (4%) | 2uL (4%) |
| MgCl2 (50mM) | 1uL (1mM) | 1uL (1mM) | 1uL (1mM) | 1uL (1mM) |
USER reaction
Performed User reaction according to standard protocol with the following parts:
AMO into psB1C3 HAO into psB1C3 cycAX into psB1C3
Sec-construct into psB1C3 TAT2-construct into psB1C3 TAT3-construct into psB1C3
ligation of pZA21::RFP with the constitutive reference promoter
Results
Gel pictures
Ran gels on yesterday's PCRs to create psB1C3 with USER endings.
Small gel:
- 1 kb ladder
- 2% DMSO, pur
- 4% DMSO, pur
- 5% DMSO, pur
- 4% DMSO, 1uL 50mM MgCl2, pur
- 4% DMSO, 2uL 50mM MgCl2, pur
- 1M Betaine, pur
- 2% DMSO, HQ
- 4% DMSO, HQ
- 5% DMSO, HQ
- 4% DMSO, 1uL, HQ
- 4% DMSO, 2uL, HQ
- 1M Betaine, HQ
- 1 kb ladder (ladder was almost empty so I put another one in the next lane)
- 1 kb ladder
big gel (samples from touch down PCR):
- 1 kb ladder
- GC, HQ
- 2% DMSO HQ
- 4% DMSO HQ
- 5% DMSO HQ
- 4% DMSO, 1uL 50mM MgCl2, HQ
- 4% DMSO, 2uL 50mM MgCl2, HQ
- 1M Betaine, HQ
- GC pur
- 2% DMSO, pur
- 4% DMSO, pur
- 5% DMSO, pur
- 4% DMSO, 1uL 50mM MgCl2, pur
- 4% DMSO, 2uL 50mM MgCl2, pur
- 1M Betaine, pur
- neg
- 1 kb ladder
Conclusion
PCR to get USER ends on pSB1C3 is substantially better with a TouchDown PCR than with a fixed annealing temperature. It does also positively affect the product yield to add 4% of DMSO and 1uL 50mM MgCl2.
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