Team:DTU-Denmark/Notebook/7 August 2013
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temperature: 53C, time: 3:00 | temperature: 53C, time: 3:00 | ||
Samples: | Samples: | ||
- | 1->SLP | + | 1->SLP + DMSO |
+ | 2 -> SLP + betaine | ||
+ | 3 -> Ref + DSMO | ||
+ | 4 -> Ref + betaine | ||
===PCR for AMO with USER endings=== | ===PCR for AMO with USER endings=== |
Revision as of 13:32, 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
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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