Team:Stockholm/6 October 2010
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+ | |||
+ | ===Sequencing results=== | ||
+ | Received sequencing results for the following samples ([[media:Sequencings_fasta_6oct.txt|fasta]]): | ||
+ | *pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 1 (VF2 & VR) | ||
+ | *pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR) | ||
+ | *pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR) | ||
+ | *pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR) | ||
+ | *pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR) | ||
+ | *pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR) | ||
+ | *pEX.nTra10⋅SOD⋅His (pEXf) | ||
+ | *pEX.nLMWP⋅SOD⋅His (pEXf) | ||
+ | |||
+ | Sequences aligned using Geneious software, showing correct sequences for all constructs. Operon constructs all had an insertion between SpeI and PstI; this will not affect expression. | ||
+ | |||
+ | ===Plasmid prep=== | ||
+ | ''Of stored pellets and ON cultures from 5/10'' | ||
+ | |||
+ | *E.Z.N.A. Plasmid Miniprep kit | ||
+ | **50 μ elution volume (elution buffer) | ||
+ | |||
+ | {|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> | ||
+ | |- | ||
+ | |pEX.nTAT⋅SOD⋅His.RBS.yCCS | ||
+ | |align="center"|107.5 | ||
+ | |align="center"|1.96 | ||
+ | |- | ||
+ | |pEX.nTra10⋅SOD⋅His.RBS.yCCS | ||
+ | |align="center"|108.7 | ||
+ | |align="center"|1.93 | ||
+ | |- | ||
+ | |pEX.nLMWP⋅SOD⋅His.RBS.yCCS | ||
+ | |align="center"|84.35 | ||
+ | |align="center"|1.97 | ||
+ | |- | ||
+ | |pSB1C3.His⋅SOD⋅cLMWP B | ||
+ | |align="center"|255.4 | ||
+ | |align="center"|1.94 | ||
+ | |- | ||
+ | |pSB1C3.His⋅SOD⋅cLMWP C | ||
+ | |align="center"|201.2 | ||
+ | |align="center"|1.94 | ||
+ | |- | ||
+ | |pSB1C3.His⋅SOD⋅cTAT D | ||
+ | |align="center"|369.5 | ||
+ | |align="center"|1.92 | ||
+ | |- | ||
+ | |pSB1C3.His⋅SOD⋅cTra10 A | ||
+ | |align="center"|188.6 | ||
+ | |align="center"|1.95 | ||
+ | |} | ||
+ | |||
+ | ===BL21 transformation=== | ||
+ | Quick transformation w/ 30 min on ice, 50 sec heat-shock. | ||
+ | *30 μl competent BL21(DE3) | ||
+ | *1 μl plasmid | ||
+ | **pEX.nTra10⋅SOD⋅His.RBS.yCCS | ||
+ | **pEX.nTAT⋅SOD⋅His.RBS.yCCS | ||
+ | **pEX.nLMWP⋅SOD⋅His.RBS.yCCS |
Revision as of 16:40, 6 October 2010
Contents |
Andreas
Sequencing results
Received sequencing results for the following samples (fasta):
- pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 1 (VF2 & VR)
- pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
- pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
- pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
- pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
- pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
- pEX.nTra10⋅SOD⋅His (pEXf)
- pEX.nLMWP⋅SOD⋅His (pEXf)
Sequences aligned using Geneious software, showing correct sequences for all constructs. Operon constructs all had an insertion between SpeI and PstI; this will not affect expression.
Plasmid prep
Of stored pellets and ON cultures from 5/10
- E.Z.N.A. Plasmid Miniprep kit
- 50 μ elution volume (elution buffer)
DNA concentration | ||
---|---|---|
Sample | Conc [ng/μl] | A260/A280 |
pEX.nTAT⋅SOD⋅His.RBS.yCCS | 107.5 | 1.96 |
pEX.nTra10⋅SOD⋅His.RBS.yCCS | 108.7 | 1.93 |
pEX.nLMWP⋅SOD⋅His.RBS.yCCS | 84.35 | 1.97 |
pSB1C3.His⋅SOD⋅cLMWP B | 255.4 | 1.94 |
pSB1C3.His⋅SOD⋅cLMWP C | 201.2 | 1.94 |
pSB1C3.His⋅SOD⋅cTAT D | 369.5 | 1.92 |
pSB1C3.His⋅SOD⋅cTra10 A | 188.6 | 1.95 |
BL21 transformation
Quick transformation w/ 30 min on ice, 50 sec heat-shock.
- 30 μl competent BL21(DE3)
- 1 μl plasmid
- pEX.nTra10⋅SOD⋅His.RBS.yCCS
- pEX.nTAT⋅SOD⋅His.RBS.yCCS
- pEX.nLMWP⋅SOD⋅His.RBS.yCCS