IGEM:IMPERIAL/2006/LabCalendar/2006-9-5
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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
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) |
J37015RS | Riboswitch | 44[[:Category:{{{1}}}|{{{1}}}]] |
J37020 | Plasmid A2 reverse primer | 56[[:Category:{{{1}}}|{{{1}}}]] |
J37023 | T3 and T7 primers (from Dr.Mann) | 39[[:Category:{{{1}}}|{{{1}}}]] for both |
J37025 | Plasmid A2 forward primer | 50[[: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