IGEM:IMPERIAL/2006/LabCalendar/2006-9-5: Difference between revisions
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*There was no discernable difference in the proteins produced by the induced and non-induced cells | *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 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. | *This does not however mean that the aiiA is wrong necessarily - testing of J37022 should clarify this. | ||
[[Image:AiiAtestexpression.JPG|500px]] | |||
==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 | |||
<br> | |||
==LoxP PCR== | ==LoxP PCR== | ||
* The small PCR fragment is not being amplified | * 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== | ==Sequencing== | ||
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| J37025||Plasmid A2 forward primer||50{{c}} | | J37025||Plasmid A2 forward primer||50{{c}} | ||
|} | |} | ||
==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 | |||
Latest revision as of 14:22, 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
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
