Team:Stockholm/23 September 2010
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==Andreas== | ==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==== | ||
+ | [[image:ColPCR_BL21_clones_23sep.png|200px|thumb|right|'''Colony PCR gel verification of BL21 clones transformed with pEX.SOD, pEX.SOD⋅His, pEX.His⋅SOD or pEX.yCCS 5.'''<br />4 μl λ; 5 μl sample.<br />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'''<br /> | ||
+ | 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. | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | !colspan="3"|DNA concentration | ||
+ | |- | ||
+ | !Sample | ||
+ | !width="60"|Conc [ng/μl] | ||
+ | !width="60"|A<sub>260</sub>/A<sub>280</sub> | ||
+ | |- | ||
+ | |pSB1C3.N-LMWP⋅SOD⋅His 1 | ||
+ | |align="center"|35.06 | ||
+ | |align="center"|1.68 | ||
+ | |- | ||
+ | |pSB1C3.N-LMWP⋅SOD⋅His 4 | ||
+ | |align="center"|46.85 | ||
+ | |align="center"|1.68 | ||
+ | |- | ||
+ | |pSB1A2.RBS.yCCS 3 | ||
+ | |align="center"|39.64 | ||
+ | |align="center"|1.59 | ||
+ | |- | ||
+ | |pSB1A2.RBS.yCCS 4 | ||
+ | |align="center"|87.01 | ||
+ | |align="center"|1.49 | ||
+ | |- | ||
+ | |pEX.N-TAT⋅SOD⋅His 3 | ||
+ | |align="center"|66.22 | ||
+ | |align="center"|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) |
Revision as of 21:06, 23 September 2010
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)