IGEM:IMPERIAL/2006/LabCalendar/2006-9-5

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*Was started with the revised protocol but after results from Western Blot came out, was abandonded
*Was started with the revised protocol but after results from Western Blot came out, was abandonded
*Is not going to be tested any more since no protein seems to be transcribed at all
*Is not going to be tested any more since no protein seems to be transcribed at all
 +
 +
==AiiA Ligation/electroporation==
 +
*Samples run on gel and adequate bands cut out.
 +
*Glass milk purified and ligated into T9 overhanging vector
 +
*Forms New J37023
 +
*Electroporated in the afternoon
 +
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==LoxP PCR==  
==LoxP PCR==  

Revision as of 05:18, 8 September 2006



TO DO:
*Testing of J37016, S01656
*Finish ligation for J37022 & electroporate (has it been finished yesterday?)
*Finish ligation for Dr. Frays aiiA into the T hanging plasmid & electroporate
*Culture J37036 & J37018 from plate in incubator for maxi & mini on Wednesday
*Culture T9002 from plate in incubator for new testing & making frozen stocks

Contents

Western Blot

  • There was no discernable difference in the proteins produced by the induced and non-induced cells
  • This indicates that there is something wrong with the mechanism involving S01656 expression, possibly its promoter
  • This does not however mean that the aiiA is wrong necessarily - testing of J37022 should clarify this.

Testing of S01656

  • Was started with the revised protocol but after results from Western Blot came out, was abandonded
  • Is not going to be tested any more since no protein seems to be transcribed at all

AiiA Ligation/electroporation

  • Samples run on gel and adequate bands cut out.
  • Glass milk purified and ligated into T9 overhanging vector
  • Forms New J37023
  • Electroporated in the afternoon


LoxP PCR

  • The small PCR fragment is not being amplified

Testing S01656 & J37016

  • Testing was not conducted as western blot indicates that there may be something wrong with the S01656 expression mechanism.
  • J37016 was being cultured for the S01656 testing. This test, too, was not finished since we are no longer carrying out testing of S01656. We have 3 good sets of data for J37016 so far.

Sequencing

Part Primer used Melting temp (Tm)
J37015RSRiboswitch44[[:Category:{{{1}}}|{{{1}}}]]
J37020Plasmid A2 reverse primer56[[:Category:{{{1}}}|{{{1}}}]]
J37023T3 and T7 primers (from Dr.Mann)39[[:Category:{{{1}}}|{{{1}}}]] for both
J37025Plasmid A2 forward primer50[[:Category:{{{1}}}|{{{1}}}]]

Culturing

  • Cultured up 4 colonies of J37036 in 110ml of LB Amp for maxi and mini
  • Cultured up 2 colonies of J37018 in 110ml of LB Amp for maxi and mini
  • Cultured up 2 colonies of T9002 in 2ml of LB Amp for tesing?
  • Cultured up J37015 in 2ml of LB Amp for testing
  • Cultured up J37016 in 2ml of LB Amp for testing
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