Team:Stockholm/4 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
===Transfer of pEX.nCPP⋅SOD⋅His to BL21===
 +
====Gel verification====
 +
''Re-run of 2/10 BL21 samples''
 +
 +
#BL21 pEX.nLMWP⋅SOD⋅His: A & B
 +
#BL21 pEX.nTra10⋅SOD⋅His: A & B
 +
 +
1 % agarose, 120 V
 +
 +
'''Expected bands:'''
 +
#744 bp
 +
#765 bp
 +
 +
'''Results'''
 +
#Both clones verified
 +
#Clone A verified; weak band for clone B
 +
 +
====ON cultures====
 +
*3 ml LB, 30 °C
 +
*# A: BL21 pEX.nLMWP⋅SOD⋅His
 +
*# 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====
 +
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

Revision as of 14:13, 4 October 2010

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Contents

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

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

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
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