Team:Cambridge/Lab book/Week 6

From 2012.igem.org

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[[File:vectorgel1.jpg|250px|thumb|Top:]]
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[[File:vectorgel1.jpg|250px|thumb|Top: Fusion, lux containing vector gel. Lanes 2-4, Vector DNA. Lane 5, +ve control. Lane 6, -ve control. Bottom: Vector gel. Lanes 1-3, Fluorescent contruct vector DNA. Lanes 4-6, Riboswitch construct vector DNA.]]
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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.
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*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.
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PCR cycle x35:  
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*PCR cycle x35:  
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* 15s Denaturing at 95 C
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:* 15s Denaturing at 95 C
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* 45s Annealing at 60 C
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:* 45s Annealing at 60 C
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* 300s Extension at 72 C
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:* 300s Extension at 72 C
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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.
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*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.
'''[[Team:Cambridge/Protocols/Gibsonassembly|Construction of riboswitch plasmid with Gibson Assembly]]'''
'''[[Team:Cambridge/Protocols/Gibsonassembly|Construction of riboswitch plasmid with Gibson Assembly]]'''

Revision as of 13:02, 2 August 2012

Week: 3 4 5 6 7

Contents

Monday

PCR of vectors


Top: Fusion, lux containing vector gel. Lanes 2-4, Vector DNA. Lane 5, +ve control. Lane 6, -ve control. Bottom: Vector gel. Lanes 1-3, Fluorescent contruct vector DNA. Lanes 4-6, Riboswitch construct vector DNA.
  • 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
  • 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