Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/02: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav) |
|||
(2 intermediate revisions by one other user not shown) | |||
Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">gBlock (DBN001) & DBN002 PCR Amplification</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">gBlock (DBN001) & DBN002 PCR Amplification; pSB1A3-GFP Restriction Digest</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}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
Line 174: | Line 174: | ||
| align="center" style="background:#f0f0f0;"|''''' | | align="center" style="background:#f0f0f0;"|''''' | ||
| align="center" style="background:#f0f0f0;"|''''' | | align="center" style="background:#f0f0f0;"|''''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''pSB1A3-GFP''' | ||
|- | |- | ||
| ||49C||50C||51C||52C||53C||54C||55C||56C||n/a | | ||49C||50C||51C||52C||53C||54C||55C||56C||n/a | ||
Line 181: | Line 181: | ||
[[Image:2015-04-03_PCR_products_part_2_annotated.png | 500px]] | [[Image:2015-04-03_PCR_products_part_2_annotated.png | 500px]] | ||
It looks like I ran the gel for too long, so the expected size fragment for the gBlock would have run into the | It looks like I ran the gel for too long, so the expected size fragment for the gBlock would have run into the pSB1A3-GFP lane. Will need to run that again. It also looks as though the pSB1A3-GFP digestion was not successful. Will need to re-try with more plasmid, as I used most of it on transfecting the cells. | ||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Latest revision as of 00:52, 27 September 2017
gBlock (DBN001) & DBN002 PCR Amplification; pSB1A3-GFP Restriction Digest | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OverviewRunning out of the gBlock ordered (DBN001), and it's not yet in a plasmid, so I'm making more by PCR amplification. gBlock PCR run details:
Run is saved as DBN20140402B on the Bio-Rad thermal cycler. DBN002 PCR Run
Run is saved as DBN20140402A on the Bio-Rad thermal cycler. pSB1A3-GFP plasmid digestion
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.
Gel Electrophoresis of DBN001, DBN002 and pSB1A3-GFP ProductsGel layout:
It looks like I ran the gel for too long, so the expected size fragment for the gBlock would have run into the pSB1A3-GFP lane. Will need to run that again. It also looks as though the pSB1A3-GFP digestion was not successful. Will need to re-try with more plasmid, as I used most of it on transfecting the cells. |