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From 2010.igem.org

Contents 1 Andreas 1.1 BL21(DE3) clone verification 1.1.1 Colony PCR 1.1.2 Gel verification 1.1.3 ON cultures 1.2 Plasmid prep 1.2.1 ON cultures 2 Johan 2.1 Cut

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

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