Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/18: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Project Introduction</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Project Introduction</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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# Isolate single colonies of stably transfected cells, grow up, and make stocks. | # Isolate single colonies of stably transfected cells, grow up, and make stocks. | ||
==DBN002 & DBN003 PCR== | |||
Primers and gBlock have already been ordered, and primers arrived yesterday. Today I made 100μM stocks of the primers and used the primers to amplify the HPK sequence (with Kozak) and the CFP sequence. | Primers and gBlock have already been ordered, and primers arrived yesterday. Today I made 100μM stocks of the primers and used the primers to amplify the HPK sequence (with Kozak) and the CFP sequence. | ||
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| align="center" style="background:#f0f0f0;"|'''Reagent''' | | align="center" style="background:#f0f0f0;"|'''Reagent''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume ( | | align="center" style="background:#f0f0f0;"|'''Volume (µL)''' | ||
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| 2x master mix||25 | | 2x master mix||25 |
Latest revision as of 00:50, 27 September 2017
Project Introduction | Main project page Next entry | |||||||||||||||||||||||||||||||||||
OverviewThe goal of this project is to change the reporter gene in a stably transfected cancer cell line. The current reporter gene is luciferase, which has poor resolution on a cellular level and requires a time-intensive assay to measure. We intend to replace the luciferase gene and promoter with cyan fluorescent protein (CFP) for rapid measurement of reporter gene activity. We'll also be replacing the luciferase TK promoter with a HPK promoter, which should enhance transcription. Here's the overall direction this project is going to take:
DBN002 & DBN003 PCRPrimers and gBlock have already been ordered, and primers arrived yesterday. Today I made 100μM stocks of the primers and used the primers to amplify the HPK sequence (with Kozak) and the CFP sequence. PCR kit used was GoTaq clear 2x Master Mix, containing polymerase, buffer, and dNTPs.
Four reactions were performed. Rxn 1 used KAH45 plasmid as the template (from BioBricks box) and DBN002 f1 and DBN002 r1 as the primers. Rxn 2 used KAH104 plasmid as the template (from BioBricks box) and DBN003 f1 and DBN003 r1 as the primers. Each reaction also had a control reaction containing primers but no template. PCR program used is named DBN031815. 30 cycles were performed.
The DBN002 primers anneal at 59°C and the DBN003 primers anneal at 68°C. |