Team:Stockholm/23 September 2010

From 2010.igem.org


Contents

Andreas

BL21(DE3) clone verification

Attempted to verify BL21 clones from 21/9 transformations for correct plasmid (pEX) and insert.

Colony PCR

  • pEX.SOD: 1 & 2
  • pEX.SOD⋅His (pEX.SH): 1 & 2
  • pEX.His⋅SOD (pEX.HS): 1 & 2
  • pEX.yCCS 5: 1 & 2
  • pEX.His⋅SOD plasmid (PC)

PCR settings according to standard colony PCR protocol.

  • Elongation time: 1:30

Gel verification

Colony PCR gel verification of BL21 clones transformed with pEX.SOD, pEX.SOD⋅His, pEX.His⋅SOD or pEX.yCCS 5.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 120 V

Expected bands

  • pEX.SOD: 678 bp
  • pEX.SOD⋅His (pEX.SH): 702 bp
  • pEX.His⋅SOD (pEX.HS): 702 bp
  • pEX.yCCS: 963 bp

Results
Very weak but correct-sized bands for all clones but pEX.yCCS 5 #1. Clones verified.

ON cultures

One verified clone of each BL21 construct was chosen for preparation of glycerol stocks:

  • 3 ml LB + 100 Amp; 30 °C
    • pEX.SOD: clone 1
    • pEX.SOD⋅His (pEX.SH): clone 1
    • pEX.His⋅SOD (pEX.HS): clone 1
    • pEX.yCCS: clone 2

Plasmid prep

From 22/9 ON cultures

Using Omega E.Z.N.A. Plasmid Miniprep kit I.

No growth in cultures pSB1K3.N-LMWP⋅SOD⋅His and pEX.N-TAT⋅SOD⋅His 4.

DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.N-LMWP⋅SOD⋅His 1 35.06 1.68
pSB1C3.N-LMWP⋅SOD⋅His 4 46.85 1.68
pSB1A2.RBS.yCCS 3 39.64 1.59
pSB1A2.RBS.yCCS 4 87.01 1.49
pEX.N-TAT⋅SOD⋅His 3 66.22 1.67

Very low DNA concentrations and low purity. Possibly something wrong with the plasmid miniprep kit. New plasmid preps will be prepared tomorrow, using brand new kit solutions.

ON cultures

  • 5 ml LB + antibiotic (100 Amp, 50 Km or 25 Cm); 37 °C 200 rpm
    • pEX.N-TAT⋅SOD⋅His 3 (100 Amp)
    • pEX.N-TAT⋅SOD⋅His 4 (100 Amp)
    • pSB1C3.N-LMWP⋅SOD⋅His 1 (25 Cm)
    • pSB1C3.N-LMWP⋅SOD⋅His 4 (25 Cm)
    • pSB1A2.RBS.yCCS 3 (100 Amp)
    • pSB1A2.RBS.yCCS 4 (100 Amp)
    • pSB1K3.N-LMWP⋅SOD⋅His 1 (50 Km)
    • pSB1K3.N-TAT⋅SOD⋅His 4 (50 Km)
    • pSB1K3.N-TAT⋅SOD⋅His 5 (50 Km)
    • pSB1K3.N-Tra10⋅SOD⋅His 5 (50 Km)

Johan

Cut

bFGF with EcoRI and AgeI

bFGF with NgoMIV and SpeI

pMA with EcoRI and NgoMIV

pMA with AgeI and SpeI

5 µl DNA

1 µl of both enzymes

2 µl 10x fastbuffer

11 µl H2O





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