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Team:DTU-Denmark/Notebook/7 August 2013
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===PCR for SPL and Ref (reference promoter)=== | ===PCR for SPL and Ref (reference promoter)=== | ||
template: pZa21 with RFP; | template: pZa21 with RFP; | ||
| + | |||
primers for SLP: 52a, 52b1; | primers for SLP: 52a, 52b1; | ||
primers for Ref: 52a, 52b2; | primers for Ref: 52a, 52b2; | ||
| - | temperature: 53C, time: 3:00 | + | |
| + | temperature: 53C, time: 3:00; | ||
| + | |||
Samples: | Samples: | ||
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4 -> Ref + betaine | 4 -> Ref + betaine | ||
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| + | 5 -> SLP Negative control + DMSO | ||
| + | |||
| + | 6 -> Ref Negative control + betaine | ||
===PCR for AMO with USER endings=== | ===PCR for AMO with USER endings=== | ||
Revision as of 13:35, 7 August 2013
7 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Kristian, Gosia, Julia, Henrike
Procedure
PCR for SPL and Ref (reference promoter)
template: pZa21 with RFP;
primers for SLP: 52a, 52b1; primers for Ref: 52a, 52b2;
temperature: 53C, time: 3:00;
Samples:
1 -> SLP + DMSO
2 -> SLP + betaine
3 -> Ref + DSMO
4 -> Ref + betaine
5 -> SLP Negative control + DMSO
6 -> Ref Negative control + betaine
PCR for AMO with USER endings
Midiprep
Purifying plasmids with high yield for sequencing of the samples cycAX, Sec2, TAT2-1, TAT3-2 and TAT3-1a.
Gel purification
Extracted from gel and purified AMO extraction fragment, araBAD promoter, Nir2 extraction fragment.
Results
Gel on yesterdays PCR
ara, SPL is from yesterdays PCR with 5% DMSO, Nir and the sample numbers is also from yesterday, here the composition can be seen.
- ara
- ara
- SPL
- SPL
- Nir 0 MgCl2
- Nir 1uL MgCl2
- Nir 5uL MgCl2
- Nir 20uL MgCl2
- 12 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, x7 polymerase
- 13 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
- 14 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
- 15 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
- 16 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
- 7 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
- 8 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
- 9 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
- 10 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
not on the gel: 11 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, Phusion polymerase
Decided to purify ara and Nir2 (from 12, 7, 8, 9 and 10) and analyse sample 11 (Nir2).
Purification gel
- 1 kb ladder
- ara
- ara
- 12
- 7
- 8
- 9
- 10
- 11
- Nir colony PCR X7
- 1 kb
lab 115
Main purpose
Who was in the lab
Ariadni, Helen
Conclusion
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