Team:Cambridge/Lab book/Week 12

From 2012.igem.org

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===Thursday (13/09/12)===
===Thursday (13/09/12)===
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'''Magnesium Riboswitch Plate-Reader Assay'''
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 +
----
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 +
*Plate reader assay set up with magnesium riboswitch construct made over the last few days.
 +
 +
*Mg2+ concentrations used:
 +
 +
:*5μM
 +
:*10μM
 +
:*20μM
 +
:*50μM
 +
:*100μM
 +
:*200μM
 +
:*500μM
 +
:*1mM
 +
:*2mM
 +
:*5mM
 +
:*10mM
 +
:*20mM
 +
 +
*IPTG concentrations used:
 +
 +
:*0.05mM
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:*0.1mM
 +
:*0.2mM
 +
:*0.5mM
 +
:*1mM
 +
:*2mM
 +
:*5mM
 +
:*10mM
===Friday (14/09/12)===
===Friday (14/09/12)===

Revision as of 03:32, 27 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.

Restriction digest of magnesium riboswitch construct plasmids

  • Colonies grown up from colonies produced from Gibson products made on Check!!! were miniprepped and plasmid DNA extracted.
  • Restriction digest performed with Sal1 and BamH1. Expected fragment sizes: 4900bp and 4500bp. If plasmid lacks insert: 4900bp and 3950bp. These two possibilities should be distinguishable.
  • Correct banding pattern seen for all colonies grown up, with both -8 and +8 construct. Shall verify identity further with colony PCR.

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)

Magnesium Riboswitch Plate-Reader Assay

  • Plate reader assay set up with magnesium riboswitch construct made over the last few days.
  • Mg2+ concentrations used:
  • 5μM
  • 10μM
  • 20μM
  • 50μM
  • 100μM
  • 200μM
  • 500μM
  • 1mM
  • 2mM
  • 5mM
  • 10mM
  • 20mM
  • IPTG concentrations used:
  • 0.05mM
  • 0.1mM
  • 0.2mM
  • 0.5mM
  • 1mM
  • 2mM
  • 5mM
  • 10mM

Friday (14/09/12)

Saturday (15/09/12)

Sunday (16/09/12)

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