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Team:DTU-Denmark/Notebook/20 August 2013
From 2013.igem.org
20 August 2013
Contents |
Lab 208
Main purpose
- PCR to linearize pZA21::RFP::araBAD
Who was in the lab
Kristian, Helen, Henrike, Julia
Procedure
PCR to linearize pZA21::RFP::araBAD
- Template: pZA21::RFP::araBAD
- Primers: 55a and 55b
- Program:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 65C | 1:00 | 36 |
| 72C | 3:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
The table shows the composition of each reaction:
| component (per reaction) | 1 | 2 | 3 | 4 | 5 | 6 | Neg |
|---|---|---|---|---|---|---|---|
| dNTPs | 1uL | 1uL | 1uL | 1uL | 1uL | 1uL | - |
| buffer (GC/HF + volume) | HF + 10uL | HF + 10uL | HF + 10uL | GC + 10uL | HF + 10uL | HF + 10uL | HF + 10uL |
| X7 polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
| MilliQ water | 29uL | 21.5uL | 31.5uL | 29uL | 31.5uL | 31.5uL | 32.5uL |
| template | 1uL | 1uL | 1uL | 1uL | 1uL | 1uL | - |
| FW primer 55a | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL |
| RV primer 55b | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL |
| DMSO (100%) | 2.5uL (5%) | - | - | 2.5uL (5%) | - | - | - |
| Betaine (5M) | - | 10uL (1M) | - | - | - | - | - |
Repeated reaction mix number 1 and ran on same program to get more backbone.
Results
Gels
loaded more samples from the screening PCR for BioBrick creation yesterday
- 1 kb ladder
- screening D12
- screening D11
- screening D10
- screening D9
- screening D8
- screening D7
- screening D6
- screening D5
- screening D4
- screening D3
- screening D2
- screening D1
- screening C12
- screening C11
- screening C10
- screening C9
- screening C8
- 1 kb ladder
- 1 kb ladder
- screening C7
- screening C6
- screening C5
- screening C4
- screening C3
- screening C2
- screening C1
- screening B12
- screening B11
- screening B10
- screening B9
- screening B8
- screening B7
- 1 kb ladder
gel on today's PCR to linearize pZA21::ara
- 1 kb ladder
- sample 1
- sample 2
- sample 3
- sample 4
- negative controll
- 1 kb ladder
Two samples were lost because the lids opened during the PCR reaction and sample evaporated.
purification gels
Pooled Nir2 and Nir1 samples for Morten Nørholm that gave bands.
- 1 kb ladder
- pZA21::RFP::AraBAD
- pZA21::RFP::AraBAD
- pZA21::RFP::AraBAD
- Nir2
- Nir2
- Nir2
- Nir1
- Nir1
- Nir1
- Nir1
- 1 kb ladder
Second purification
- 1 kb ladder
- pZA21::RFP::AraBAD, sample 1 (from todays PCR)
- Nir1
- Nir1
- Nir2
- Nir2
- Nir2
- 1 kb ladder
Conclusion
Something is definitely wrong with our gels. It seems the gradient PCR did not give the right product. Amplification of the linearized pZA21::ara worked.
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