IGEM:MIT/2006/Notebook/2006-8-10: Difference between revisions
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==to do== | ==to do== | ||
#GC work with new smell samples | #GC work with new smell samples -'''rescheduled for 8/11''' | ||
#Miniprep all LCs/glycerols | #Miniprep all LCs/make appropriate glycerols/sequence -'''done''' | ||
#*Make 10 biobrick named glycerols and send for sequencing | #*Remember to Make 10 biobrick named glycerols and send for sequencing -'''done''' | ||
#Make LCs of IK transformants | #Make LCs of IK transformants in pm (overnight smell test and regular batch for glycerols/am smell test) -'''done''' | ||
#PCR pchBA with 5% Betane -'''done''' | |||
#PCR pchBA with 5% Betane | #site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -'''done''' | ||
#site-directed mutagenesis on banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers | |||
==PCR of pchBA== | |||
...it worked! The following showed bands on the gel and are currently in the fridge (pcr cleaned up): | |||
*pchBA pcr'ed w/ new forward and reverse primers, with 2μL betaine added to pcr rxn [53.3 ng/μL] | |||
*pchBA pcr'ed w/ new forward and reverse primers, with 4μL betaine added to pcr rxn [9.9 ng/μL] | |||
*pchBA pcr'ed w/ new forward and reverse primers, with a 96deg (instead of 94deg) denature step...we stopped the reaction on cycle 21 out of 30 b/c we needed the block for mutagenesis [17.8 ng/μL] | |||
*pchA pcr'ed w/ new reverse primer (and the old forward one), with 2 μL betaine added to pcr rxn [64.2 ng/μL] | |||
next steps: biobrick, do the pchA mutagenesis, hook it up to terminator, RBS, promoter! | |||
==Indole KO Strain== | ==Indole KO Strain== | ||
The following table summarizes the results of the indole KO transformations. | |||
{| border="1" cellpadding="5" | |||
!| | |||
!|# of Transformants | |||
!|Notes about colonies | |||
|- | |||
|'''pUC18''' | |||
|lots | |||
|essentially uniform in size (though denser areas contain smaller colonies); well spread across plate | |||
|- | |||
|'''J45100''' | |||
|~110 | |||
|many tended to grow in clusters; range of sizes from very small to large (though large colonies may be more than one) | |||
|- | |||
|'''J45120''' | |||
|~70 | |||
|not as many clusters; the colonies are huge, in general (a few small ones, though) | |||
|- | |||
|'''J45170''' | |||
|~100 | |||
|not as many clusters; the colonies are huge, in general (a few small ones, though) | |||
|- | |||
|'''J45200''' | |||
|~110 | |||
|range of sizes from very small to large; some clusters | |||
|- | |||
|'''J45220''' | |||
|~50 | |||
|a few small colonies but most are huge; few clusters | |||
|} | |||
==Mutagenesis== | |||
#Held off on BAT2 because of Spe1 site | |||
#Regrew THI3 culture because not enough cells | |||
#Mutagenesis on 4 ATF1 (200,220,250,270) | |||
#Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis | |||
Latest revision as of 15:51, 10 August 2006
to do
- GC work with new smell samples -rescheduled for 8/11
- Miniprep all LCs/make appropriate glycerols/sequence -done
- Remember to Make 10 biobrick named glycerols and send for sequencing -done
- Make LCs of IK transformants in pm (overnight smell test and regular batch for glycerols/am smell test) -done
- PCR pchBA with 5% Betane -done
- site-directed mutagenesis on ATF1 in banana generating device (with both RBS30 and RBS32), THI3 in TOPO vector with NEW mut primers -done
PCR of pchBA
...it worked! The following showed bands on the gel and are currently in the fridge (pcr cleaned up):
- pchBA pcr'ed w/ new forward and reverse primers, with 2μL betaine added to pcr rxn [53.3 ng/μL]
- pchBA pcr'ed w/ new forward and reverse primers, with 4μL betaine added to pcr rxn [9.9 ng/μL]
- pchBA pcr'ed w/ new forward and reverse primers, with a 96deg (instead of 94deg) denature step...we stopped the reaction on cycle 21 out of 30 b/c we needed the block for mutagenesis [17.8 ng/μL]
- pchA pcr'ed w/ new reverse primer (and the old forward one), with 2 μL betaine added to pcr rxn [64.2 ng/μL]
next steps: biobrick, do the pchA mutagenesis, hook it up to terminator, RBS, promoter!
Indole KO Strain
The following table summarizes the results of the indole KO transformations.
| # of Transformants | Notes about colonies | |
|---|---|---|
| pUC18 | lots | essentially uniform in size (though denser areas contain smaller colonies); well spread across plate |
| J45100 | ~110 | many tended to grow in clusters; range of sizes from very small to large (though large colonies may be more than one) |
| J45120 | ~70 | not as many clusters; the colonies are huge, in general (a few small ones, though) |
| J45170 | ~100 | not as many clusters; the colonies are huge, in general (a few small ones, though) |
| J45200 | ~110 | range of sizes from very small to large; some clusters |
| J45220 | ~50 | a few small colonies but most are huge; few clusters |
Mutagenesis
- Held off on BAT2 because of Spe1 site
- Regrew THI3 culture because not enough cells
- Mutagenesis on 4 ATF1 (200,220,250,270)
- Did PNK step http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends and mutagenesis protocl from http://openwetware.org/wiki/Knight:Site-directed_mutagenesis
