Team:Stockholm/28 September 2010

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==Andreas==
==Andreas==
===Assembly and cloning===
===Assembly and cloning===
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{{Stockholm/Footer}}

Latest revision as of 11:09, 26 October 2010

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Contents

Andreas

Assembly and cloning

Gel verification

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

Re-run of 27/9 colony PCRs.

Gel 1
1 % agarose, 110 V

Gel 2
0.8 % agarose, 90 V

Expected bands

  1. pEX.N-LMWP⋅SOD⋅His (K): 744 bp
  2. pEX.N-TAT⋅SOD⋅His: 735 bp
  3. pEX.N-Tra10⋅SOD⋅His: 765 bp
  4. pEX.N-LMWP⋅SOD⋅His (C): 744 bp
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS: 1636 bp
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS: 1666 bp
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  9. BL21 pEX.N-TAT⋅SOD⋅His 3: 735 bp
  10. BL21 pEX.N-TAT⋅SOD⋅His 4: 735 bp

Results
Listing clones of seemingly correct sizes, thereby potentially correct constructs:

  1. 1
  2. 1 & 2
  3. 1 & 2
  4. No correct clones
  5. 2, 3 & 4
  6. 1, 2 & 3
  7. 1, 2, 3 & 4
  8. No correct clones
  9. 1 & 2
  10. No correct clones

For further verification constructs should be sent for sequencing. However, we will await the sequencing results from samples sent 27/9, as they were also used for building these new assemblies.

ON cultures

Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)

  • 5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 °C, 220 rpm
1. 1
2. 1
3. 1
4. -
5. 2 & 3
6. 2 & 3
7. 1 & 2
8. -
  • 3 ml LB + Amp 100; 30 °C
9. 1



Mimmi

Gel for Andreas colony-PCR

  • Make two gels, one 1% and one 0.8% agarose
  • Load samples in number order for Andreas to analyze



Over expression

  • Start up cultures
DNA
pEX.SOD
pEX.yCCS
pEX.SOD.his
pEX.his.SOD
    • 10ml LBAMP + 100µl old ON culture
  • At OD=0.6 (3h) add 1mM IPTG
    • Take sample at 0h and 3h
      • 1ml culture
      • spinn down cells
      • remove LB
      • add 100µl sample buffer
      • freeze
      • heat to 95°C for 10min

Nina

Colony PCR screen

I performed a PCR screen on the colonies from yesterday's transformation.

pEX Master Mix 15 tubes:

  • MgCl2 7.5 ul
  • phusion buffer 5X 75 ul
  • dNTP 7.5 ul
  • PrimerF 22.5 ul
  • PrimerR 22.5 ul
  • polymerase 7.5 ul
  • H2O 225 ul

Add 24 ul/PCR tube

pMa Master Mix 10 tubes:

  • MgCl2 5 ul
  • phusion buffer 5X 50 ul
  • dNTP 5 ul
  • PrimerF 15 ul
  • PrimerR 15 ul
  • polymerase 5 ul
  • H2O 150 ul

Add 24 ul/PCR tube

Bankvector C Master Mix 25 tubes:

  • MgCl2 12.5 ul
  • phusion buffer 5X 125 ul
  • dNTP 12.5 ul
  • PrimerF 37.5 ul
  • PrimerR 37.5 ul
  • polymerase 12.5 ul
  • H2O 375 ul

Add 24 ul/PCR tube





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

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