Team:Stockholm/2 October 2010

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Contents

Andreas

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

Continued from 30/8 transformations

Colony PCR

  1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
  2. BL21 pEX.nTra10⋅SOD⋅His: A & B
  3. pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
  4. pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
  5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
  7. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
  8. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

Standard colony PCR settings.

  • Elongation time: 2:00

Gel verification

Colony PCR gel verification of BL21 pEX.nCPP*SOD*His constructs and Top10 pEX.nCPP*SOD*His.RBS.yCCS operon constructs.
4 μl λ; 4 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

  1. 744 bp
  2. 765 bp
  3. 1523 bp
  4. 1523 bp
  5. 1553 bp
  6. 1553 bp
  7. 1532 bp
  8. 1532 bp

Results
Ran gel a little too long too see correct clones of constructs 1 and 2. For the other it looks like many clones have double inserts, probably a result of too high insert:vector ratio during ligation.

Confirmed clones:

  1. -
  2. -
  3. A
  4. B
  5. -
  6. -
  7. B
  8. B

These will be saved for later.

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