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 for P.aeruginosa-Measuring the production of N2O from Nitrite NO2- anaerobically

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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