Haynes Lab:Notebook/CRISPR Editing/2014/07/19: Difference between revisions
Rene M Davis (talk | contribs) |
(fix raw html notebook nav) |
||
(4 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==07//2014== | ==07/19/2014== | ||
'''QPCR of genomic DNA isolated from KAH154 U20S cells''' | '''QPCR of genomic DNA isolated from KAH154 U20S cells''' | ||
<br><br> | <br><br> | ||
Line 31: | Line 31: | ||
| Rxn 8||no template||GAPDH - primers from KAH||3 | | Rxn 8||no template||GAPDH - primers from KAH||3 | ||
|} | |} | ||
''Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.'' | |||
<br><br> | <br><br> | ||
[[Image: 14.07.19 qPCR plate layout.png| 500px | plate layout]] | [[Image: 14.07.19 qPCR plate layout.png| 500px | plate layout]] | ||
Line 62: | Line 62: | ||
#untreated KAH154 U20S cells | #untreated KAH154 U20S cells | ||
#no template | #no template | ||
<br> | |||
10ng of genomic DNA used for each qPCR reaction | |||
<br> | <br> | ||
Template Master Mix | Template Master Mix | ||
Line 108: | Line 110: | ||
#GAPDH had a really late Cp and a lower melting temperature. Will test some from KAH's set here: http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/10/17 | #GAPDH had a really late Cp and a lower melting temperature. Will test some from KAH's set here: http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/10/17 | ||
<br><br> | <br><br> | ||
Ran the PCR products from yesterday on a gel. Was hard to see and the ladder wasn't visible but it looked like there were no bands from Target 1 - primers flanking stop codon insertion site at 72bp and one band in each lane from Target 2 - primers flanking stop codon insertion site at 162bp and Target 3 - primers flanking stop codon insertion site at 255bp | |||
<br><br> | |||
'''Microscopy on CRISPR'd KAH154 U20S cells''' | |||
<br> | |||
They were all super red.<br><br> | |||
'''Flow cytometry on CRISPR'd KAH154 U20S cells''' | |||
<br> | |||
Samples | |||
#No transfection | |||
#Water transfection | |||
#pX330 gRNA 5-1 | |||
#pX330 gRNA 5-4 | |||
#pX330 gRNA 14-2 | |||
#pX330 gRNA 14-3 | |||
<br> | |||
Harvesting the cells | |||
#Removed media | |||
#Washed with PBS | |||
#Added 0.5mL of trypsin to each well | |||
#Incubated at RT for 8 minutes | |||
#Added 1.0mL media | |||
#Loosened attached cells by pipetting. Got most of the cells up | |||
#Collect the cells in growth medium and transfer to 15 mL conical tubes. | |||
#Pellet the cells at room temperature at 1000 rpm for 3 min. | |||
#Aspirate off the growth medium, but leave enough to cover the pellets. Flick each tube to break up the pellets. Chill the cells on ice. | |||
#Wash the cells 2x with cold FACS buffer: Add 1 mL cold FACS buffer to each sample. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellets. Flick each tube to break up the pellets. Repeat this process. | |||
<br> | |||
Put cells on ice, moved to the bench | |||
<br> | |||
#Resuspend each cell sample (disrupted cell pellet) in 500 μL of cold FACS Buffer. Using a 1000 μL micropipette, forced each 500 μL of cells through a separate, labeled, clean FACS tube strainer cap. Keep the samples on ice. | |||
<br> | |||
Ran them through the flow cytometer. Didn't analyze the data yet. | |||
<!-- ##### 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 00:07, 27 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
07/19/2014QPCR of genomic DNA isolated from KAH154 U20S cells
Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.
Gene Target (primer) master mixes Gene Targets: 4 tubes
|