Team:Cambridge/Lab book/Week 6

From 2012.igem.org

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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.
*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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'''[[Team:Cambridge/Protocols/GelElectrophoresis|Separation of vector DNA]]'''
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----
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*Positive control produced no band. No primer smear - primers may not be in mix for some reason.
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*Realized correct lux vector template was not added during PCR preparation, consequently no amplification occured. Still appears to be a primer smear.
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*Fluorescent construct produced several bands. Appears to be due to mis-priming during PCR. Either changing the primers or raising the annealing temperature should solve this problem, but may mean that we have to do this PCR separately.
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*Riboswitch vector also failed. However, the extraction from the previous PCR run may have worked. We will try producing a functional plasmid with this extraction before running this PCR again.
'''[[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 13:24, 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.

Separation of vector DNA

  • Positive control produced no band. No primer smear - primers may not be in mix for some reason.
  • Realized correct lux vector template was not added during PCR preparation, consequently no amplification occured. Still appears to be a primer smear.
  • Fluorescent construct produced several bands. Appears to be due to mis-priming during PCR. Either changing the primers or raising the annealing temperature should solve this problem, but may mean that we have to do this PCR separately.
  • Riboswitch vector also failed. However, the extraction from the previous PCR run may have worked. We will try producing a functional plasmid with this extraction before running this PCR again.

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

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