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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

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)