Haynes Lab:Notebook/CRISPR Editing/2014/07/19: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
||
Line 7: | Line 7: | ||
<!-- ##### 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//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 | |||
|} | |||
<br><br> | |||
''Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.'' | |||
<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> | |||
<!-- ##### 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. ##### --> |
Revision as of 16:24, 20 July 2014
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
07//2014QPCR of genomic DNA isolated from KAH154 U20S cells
Gene Target (primer) master mixes Gene Targets: 4 tubes
|