Team:Stockholm/4 October 2010

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(Difference between revisions)
(Andreas)
(Andreas)
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===Transfer of pEX.nCPP⋅SOD⋅His to BL21===
===Transfer of pEX.nCPP⋅SOD⋅His to BL21===
====Gel verification====
====Gel verification====
 +
[[image:ColPCR_BL21CPP_4oct.png|200px|thumb|right|'''Colony PCR gel verification of BL21 clones carrying pEX.nLMWP&sdot;SOD&sdot;His (1) and pEX.nTra10&sdot;SOD&sdot;His (2) plasmids.'''<br />4 &mu;l &lambda;; 5 &mu;l sample;<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
''Re-run of 2/10 BL21 samples''
''Re-run of 2/10 BL21 samples''
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====Gel verification====
====Gel verification====
 +
[[image:ColPCR_CPP-SH-Ry_operon_4oct.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.nCPP&sdot;SOD&sdot;His.RBS.yCCS operon clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample;<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
0.8 % agarose, 100 V
0.8 % agarose, 100 V

Revision as of 15:11, 4 October 2010


Contents

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 2/10 BL21 samples

  1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
  2. BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

  1. 744 bp
  2. 765 bp

Results

  1. Both clones verified
  2. Clone A verified; weak band for clone B

ON cultures

  • 3 ml LB, 30 °C
    1. A: BL21 pEX.nLMWP⋅SOD⋅His
    2. A: BL21 pEX.nTra10⋅SOD⋅His

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

  • 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  • 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

  • Elongation time: 2:00

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

  • 5. 1553 bp
  • 6. 1553 bp

Results

  • 5. Relevant band for clone B; too large insert (double?) for clone A.
  • 6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

  • 5 ml LB, 37 °C, 250 rpm
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

  • pSB1A3
  • pSB1C3
  • pSB1K3
  • pSB1AC3
  • pSB1AK3