Team:Stockholm/23 September 2010

From 2010.igem.org

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)