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
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
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