Team:Stockholm/4 October 2010

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*pSB1AC3
*pSB1AC3
*pSB1AK3
*pSB1AK3
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===Assembly of His⋅SOD⋅cCPP constructs===
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''Continued from 1/10''
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Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.
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====Ligations====
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*Vectors:
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*#Dig pSB1C3.cTra10 E+N
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*#Dig pSB1C3.cTAT E+N
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*#Dig pSB1C3.cLMWP E+N
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*Insert: Dig pMA.His⋅SOD E+A
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{|border="1" cellpadding="1" cellspacing="0"
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| 
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!width="50"|1
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!width="50"|2
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!width="50"|3
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|-
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|10X T4 Ligase buffer
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|-
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|Vector DNA
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|Insert DNA
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|align="center"|5
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|align="center"|5
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|align="center"|5
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|-
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|dH<sub>2</sub>O
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|align="center"|11
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|align="center"|11
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|align="center"|11
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|-
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|T4 DNA ligase
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|&nbsp;
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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|}
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*Incubation: 22 &deg;C, 15 min
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====Transformations====
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#pSB1C3.His&sdot;SOD&sdot;cTra10
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#pSB1C3.His&sdot;SOD&sdot;cTAT
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#pSB1C3.His&sdot;SOD&sdot;cLMWP
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*Standard transformation
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**1 &mu;l
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**Cm 25

Revision as of 12:22, 5 October 2010

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Contents

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 2/10 BL21 samples

  1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
  2. BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

  1. 744 bp
  2. 765 bp

Results

  1. Both clones verified
  2. Clone A verified; weak band for clone B

ON cultures

  • 3 ml LB, 30 °C
    1. A: BL21 pEX.nLMWP⋅SOD⋅His
    2. A: BL21 pEX.nTra10⋅SOD⋅His

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

  • 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  • 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

  • Elongation time: 2:00

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

  • 5. 1553 bp
  • 6. 1553 bp

Results

  • 5. Relevant band for clone B; too large insert (double?) for clone A.
  • 6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

  • 5 ml LB, 37 °C, 250 rpm
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

  • pSB1A3
  • pSB1C3
  • pSB1K3
  • pSB1AC3
  • pSB1AK3

Assembly of His⋅SOD⋅cCPP constructs

Continued from 1/10

Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.

Ligations

  • Vectors:
    1. Dig pSB1C3.cTra10 E+N
    2. Dig pSB1C3.cTAT E+N
    3. Dig pSB1C3.cLMWP E+N
  • Insert: Dig pMA.His⋅SOD E+A
  1 2 3
10X T4 Ligase buffer 2 2 2
Vector DNA 1 1 1
Insert DNA 5 5 5
dH2O 11 11 11
T4 DNA ligase 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformations

  1. pSB1C3.His⋅SOD⋅cTra10
  2. pSB1C3.His⋅SOD⋅cTAT
  3. pSB1C3.His⋅SOD⋅cLMWP
  • Standard transformation
    • 1 μl
    • Cm 25
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