Team:DTU-Denmark/Notebook/7 August 2013

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(Difference between revisions)
(Procedure)
(PCR for SPL and Ref (reference promoter))
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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
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 +
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

2013-08-07 ara nir2.jpg

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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