Haynes Lab:Notebook/CRISPR Editing/2014/07/19: Difference between revisions
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==07//2014== | ==07/19/2014== | ||
'''QPCR of genomic DNA isolated from KAH154 U20S cells''' | |||
<br><br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Template DNA''' | |||
| align="center" style="background:#f0f0f0;"|'''Gene Target (primers)''' | |||
| align="center" style="background:#f0f0f0;"|'''Technical Replicates''' | |||
|- | |||
| Rxn 1||KAH154 U20S genomic DNA||Target 1 - primers flanking stop codon insertion site at 72bp||3 | |||
|- | |||
| Rxn 2||KAH154 U20S genomic DNA||Target 2 - primers flanking stop codon insertion site at 162bp||3 | |||
|- | |||
| Rxn 3||KAH154 U20S genomic DNA||Target 3 - primers flanking stop codon insertion site at 255bp||3 | |||
|- | |||
| Rxn 4||KAH154 U20S genomic DNA||GAPDH - primers from KAH||3 | |||
|- | |||
| Rxn 5||no template||Target 1 - primers flanking stop codon insertion site at 72bp||3 | |||
|- | |||
| Rxn 6||no template||Target 2 - primers flanking stop codon insertion site at 162bp||3 | |||
|- | |||
| Rxn 7||no template||Target 3 - primers flanking stop codon insertion site at 255bp||3 | |||
|- | |||
| Rxn 8||no template||GAPDH - primers from KAH||3 | |||
|} | |||
[[Image: | ''Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.'' | ||
<br><br> | |||
[[Image: 14.07.19 qPCR plate layout.png| 500px | plate layout]] | |||
'''Gene Target (primer) master mixes''' | |||
Gene Targets: 4 tubes | |||
#Target 1 - primers flanking stop codon insertion site at 72bp | |||
#Target 2 - primers flanking stop codon insertion site at 162bp | |||
#Target 3 - primers flanking stop codon insertion site at 255bp | |||
#GAPDH - primers from KAH | |||
<br> | |||
Gene Target Master Mix | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''(Single well)''' | |||
| align="center" style="background:#f0f0f0;"|'''x6.5''' | |||
|- | |||
| 2x SYBR Green Master Mix||(7.5 µL)||48.75 | |||
|- | |||
| 750 nM F/R primer mix||(3.0 µL)||19.5 | |||
|- | |||
| Total vol.||(10.5 µL)||68.25 | |||
|} | |||
<br> | |||
'''Genomic DNA Templates''' | |||
<br> | |||
Templates: 2 tubes | |||
#untreated KAH154 U20S cells | |||
#no template | |||
<br> | |||
Template Master Mix | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''(Single well)''' | |||
| align="center" style="background:#f0f0f0;"|'''x12.5''' | |||
|- | |||
| DNA or PCR H2O*||(0.067 µL)||0.8375 | |||
|- | |||
| PCR H2O||(4.433 µL)||55.4125 | |||
|- | |||
| Total vol.||(4.5 µL)||56.25 | |||
|} | |||
<br><br> | |||
'''Loading the 96-well plate''' | |||
#Pipette 13.5 µL of the appropriate Template master mix into the first well of each 3-well group. | |||
#Add 31.5 µL of the appropriate Gene Target master mix each Template mix in the first well of each 3-well group. After each addition, mix by gently pipetting up and down 3 - 5 times without making bubbles. | |||
#Transfer 15 µL of solution from the first well in each 3-well group to the second and third wells (e.g., A1 into A2, and A3). Using a fresh pipette tip for each 3-well group, to do the same for A4-6, A7-8, etc. | |||
#Seal the plate with clear film. | |||
<br><br> | |||
Ran on the Haynes lab qPCR machine with melt curve<br><br> | |||
'''Results''' | |||
<br> | |||
No template wells did not show any significant fluorescence or peaks on melt curve <br> | |||
KAH154 U20S genomic DNA | |||
''finish table'' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Primer set''' | |||
| align="center" style="background:#f0f0f0;"|'''Cp''' | |||
| align="center" style="background:#f0f0f0;"|'''Melt curve Tm''' | |||
| align="center" style="background:#f0f0f0;"|'''Melt curve area''' | |||
|- | |||
| Target 1 - primers flanking stop codon insertion site at 72bp|||||| | |||
|- | |||
| Target 2 - primers flanking stop codon insertion site at 162bp|||||| | |||
|- | |||
| Target 3 - primers flanking stop codon insertion site at 255bp|||||| | |||
|- | |||
| GAPDH - primers from KAH|||||| | |||
|} | |||
<br> | |||
'''Conclusions''' | |||
#Order new primers for Target 1 | |||
#Targets 2 and 3 work well | |||
#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> | |||
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. | |||
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Revision as of 17:05, 20 July 2014
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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
|