Team:Stockholm/12 October 2010

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(Difference between revisions)
(→Andreas)
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#Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
#Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
#All clones correct.
#All clones correct.
 +
 +
====ON cultures====
 +
*5 ml LB + Cm 25; 37 °C, 225 rpm
 +
**1C, 2C, 3C, 4C, 5C
 +
 +
===BL21 transformation and growth test===
 +
Testing BL21 cells with/without CPP expression by transforming and inducing with IPTG.
 +
 +
*50 μl 0.1 M IPTG
 +
*40 μl competent cells
 +
**pEX.SOD⋅His
 +
**pEX.nTra10⋅SOD⋅His
 +
**pEX.nTAT⋅SOD⋅His
 +
**pEX.nLMWP⋅SOD⋅His

Revision as of 15:32, 14 October 2010

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Contents

Andreas

Removal of insertion in BioBrick suffixes

Transformation results

Good colony yield on all plates. A few red colonies discovered on all plates, indicating that some partly digested pSB1C3 vector, still containing the RFP insert, had been excised during gel extraction.

Colony PCR

From 11/10 transformations'

  1. pSB1C3.IgGp A-C
  2. pSB1C3.bFGF A-C
  3. pSB1C3.ProtA A-C
  4. pSB1C3.yCCS A-C
  5. pSB1C3.SOD A-C
  • Standard colony PCR settings
    • 1:15 elongation time

Gel verification

Gel 1. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Gel 2. Colony PCR gel verification of cloned with insertion removed.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 50 bp DNA ladder.

Gel 1: 1 % agarose, 140 V
Gel 2: 1 % agarose, 160 V

Expected bands

  1. pSB1C3.IgGp: 1262 bp
  2. pSB1C3.bFGF: 794 bp
  3. pSB1C3.ProtA: 506 bp
  4. pSB1C3.yCCS: 1076 bp
  5. pSB1C3.SOD: 791 bp

Results

  1. All clones correct.
  2. All clones correct.
  3. All clones correct.
  4. Correct size for clone C, weak band for clone B. Clone A probably carrying an RFP insert.
  5. All clones correct.

ON cultures

  • 5 ml LB + Cm 25; 37 °C, 225 rpm
    • 1C, 2C, 3C, 4C, 5C

BL21 transformation and growth test

Testing BL21 cells with/without CPP expression by transforming and inducing with IPTG.

  • 50 μl 0.1 M IPTG
  • 40 μl competent cells
    • pEX.SOD⋅His
    • pEX.nTra10⋅SOD⋅His
    • pEX.nTAT⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His
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