Team:Cambridge/Lab book/Week 6

From 2012.igem.org

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===Wednesday===
===Wednesday===
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'''Mg2+ Riboswitch'''
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----
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*Successful colonies produced from transformations two days ago streaked out onto chloramphenicol (5 μg/ml) containing plates.
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*Colonies also grown up in 10ml of medium A for use with plate reader later.
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'''[[Team:Cambridge/Protocols/PCRProtocol|PCR of vector DNA]]'''
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----
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*Standard PCR rerun, this time splitting fluorescent vector into two separate sections, one of 3kb and one of 4.5kb.
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*New primers used:
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:*Fragment 1 reverse:
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:*Fragment 2 forward:
===Thursday===
===Thursday===

Revision as of 12:47, 2 August 2012

Week: 3 4 5 6 7

Contents

Monday

PCR of vectors

Not all products were obtained during Friday's PCR. Most of these missing products were large vector backbones. They are being run again, with a much longer extension time of 300s. If that fails, primers will be ordered to split the vectors into manageable chunks, and the PCR reattempted when they arrive.

PCR cycle x35:

  • 15s Denaturing at 95 C
  • 45s Annealing at 60 C
  • 300s Extension at 72 C

The remaining stray product had a slightly tricky secondary structure at the 3' end. It will be run at a series of annealing temperatures in a PCR machine capable of a temperature gradient.

Construction of riboswitch plasmid with Gibson Assembly

  • DNA from lanes 27+28, 27+29, 27+30 from gels run on Friday fused together with Gibson assembly to produce riboswitch construct. This does not have replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.
  • DNA from lanes 22,23 and 24 fused with riboswitch DNA produced two weeks ago to produce riboswitch construct. This has replacement of the first 8 codons of lac I with the 8 codons native to the gene downstream of the riboswitch.

Transformation of Bacillus with riboswitch construct

  • Plasmids made by Gibson transformed into bacillus cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates.

Tuesday

Transformation of Bacillus with riboswitch construct

  • Plasmids made by Gibson transformed into bacillus cells made two weeks ago and transformants plated out on 5μg/ml chloramphenicol plates.

Transformation of E.coli with riboswitch construct

  • Plasmids made by Gibson transformed into TOP10 e.coli cells and transformants plated out on 100μg/ml ampicillin plates.

Wednesday

Mg2+ Riboswitch

  • Successful colonies produced from transformations two days ago streaked out onto chloramphenicol (5 μg/ml) containing plates.
  • Colonies also grown up in 10ml of medium A for use with plate reader later.

PCR of vector DNA

  • Standard PCR rerun, this time splitting fluorescent vector into two separate sections, one of 3kb and one of 4.5kb.
  • New primers used:
  • Fragment 1 reverse:
  • Fragment 2 forward:

Thursday

Friday

Retrieved from "http://2012.igem.org/Team:Cambridge/Lab_book/Week_6"