Team:DTU-Denmark/Notebook/7 August 2013
From 2013.igem.org
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 and Nir2 extraction fragment. Nir2 was treated according to protocol for fragments bigger 4kb, the others as fragments between 100bp and 4kb.
USER reaction
Performed USER ligation on araBAD inducible promoter and pZA21 without native promoter containing GFP Sf respective RFP following the standard protocol (I assume).
more PCR
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
Run experiment 2: "Measuring the production of N2O from Nitrite NO2-
anaerobically in order to characterize the behavior of P.aeruginosa.
Who was in the lab
Ariadni, Helen
Procedure
Following the protocol "Experiment 2: Measuring production of N2O from Nitrite NO2- anaerobically"
Conclusion
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