Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/25: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==Colony PCR Results== | ==Colony PCR Results== | ||
[[Image:2015-11-25_DBN008_pJET_gel.tif]] | [[Image:2015-11-25_DBN008_pJET_gel.tif | 250px]] | ||
No insert detected in any of the 8 colonies picked. Going to assume this was a failure. Not proceeding to sequencing. | No insert detected in any of the 8 colonies picked. Going to assume this was a failure. Not proceeding to sequencing. |
Latest revision as of 01:21, 27 September 2017
Today's project is... | Main project page Previous entry Next entry | |||||||||||||
OverviewLigation results, colony PCR & results, possibly submitting samples for sequencing. Ligation ResultsTreatment (LCR product & pJET): ~25 colonies Negative control (pJET only): 4 colonies Looks like the ligation worked. Will pick 8 colonies and proceed with colony PCR. Colony PCRPrepare PCR tubes with the following mixture per tube:
Label tubes 1-8. For each tube, pick 1 colony from the ligation plate using a 20 µL pipette tip and place the tip directly into the PCR tube. Allow the tubes & tips to incubate for 10 minutes, then streak a labeled LB-Amp plate with the tips. Set up PCR using the lab thermal cycler with annealing temperature of 58°C. Colony PCR ResultsNo insert detected in any of the 8 colonies picked. Going to assume this was a failure. Not proceeding to sequencing. |