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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