Team:Cambridge/Lab book/Week 12

From 2012.igem.org

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===Tuesday (11/09/12)===
===Tuesday (11/09/12)===
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'''[[Team:Cambridge/Protocols/PCRProtocol|PCR of biobrick backbone]]'''
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----
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*Retried PCR of backbone for +8 magnesium riboswitch biobrick and fluoride biobrick at 60 °C and 64 °C.
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*Verification gel showed that no bands of the appropriate size came out. Once more, only primer dimers produced.
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*Production of primer dimers at these temperatures causes us to doubt that a successful PCR will be carried out at these temperatures. We may have to switch to ordinary ligation to construct our biobricks for submission.
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'''[[Team:Cambridge/Protocols/PCRcolony|Colony PCR of biobrick containing colonies]]'''
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----
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*Gibson assembly of biobrick plasmid yesterday produced a few colonies. These were tested with the standard sequencing primers to check if an insert of the correct size had been introduced into the bacteria.
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*Gel showed that fragments of the appropriate size were produced for one of the -8 biobricks and one of the -8 colonies. These will be grown up, miniprepped and sent for sequencing.
===Wednesday (12/09/12)===
===Wednesday (12/09/12)===

Revision as of 00:44, 17 September 2012

Week: 3 4 5 6 7 8 9 10 11 12 13 14

Contents

Monday (10/09/12)

PCR of biobrick vector DNA

  • Attempted to run PCR of the backbone for the biobricks once more. Settings: Annealing temperature: 56°C, elongation time: 30secs.
  • After running on gel, saw that while the -8 vector amplification appears to be producing some correctly sized (~2kb) bands, the other two only seem to be forming many primer dimers. Analysis of the sequence of the prefix and suffix revealed a CG palindrome that appears to be causing self annealing. Inserts are not affected, as they have the palindrome at the 5' end of the primer.
  • Will retry at higher temperatures tomorrow.

Gibson assembly of biobricks

  • Attempted Gibson assembly for the -8 magnesium riboswitch biobrick with the backbone fragments produced earlier today and the insert fragments produced yesterday.

Transformation of e.coli with Gibson products

  • E.coli cells transformed with Gibson products produced earlier today.
  • Transformants plated out on 25μ/ml chloramphenicol plates.

Tuesday (11/09/12)

PCR of biobrick backbone

  • Retried PCR of backbone for +8 magnesium riboswitch biobrick and fluoride biobrick at 60 °C and 64 °C.
  • Verification gel showed that no bands of the appropriate size came out. Once more, only primer dimers produced.
  • Production of primer dimers at these temperatures causes us to doubt that a successful PCR will be carried out at these temperatures. We may have to switch to ordinary ligation to construct our biobricks for submission.

Colony PCR of biobrick containing colonies

  • Gibson assembly of biobrick plasmid yesterday produced a few colonies. These were tested with the standard sequencing primers to check if an insert of the correct size had been introduced into the bacteria.
  • Gel showed that fragments of the appropriate size were produced for one of the -8 biobricks and one of the -8 colonies. These will be grown up, miniprepped and sent for sequencing.

Wednesday (12/09/12)

Thursday (13/09/12)

Friday (14/09/12)

Saturday (15/09/12)

Sunday (16/09/12)

Retrieved from "http://2012.igem.org/Team:Cambridge/Lab_book/Week_12"