07/19/2014
QPCR of genomic DNA isolated from KAH154 U20S cells
'
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Template DNA
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Gene Target (primers)
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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
|
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
- 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
Gene Target Master Mix
Reagent
|
(Single well)
|
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
|
Genomic DNA Templates
Templates: 2 tubes
- untreated KAH154 U20S cells
- no template
Template Master Mix
Reagent
|
(Single well)
|
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
|
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.
Ran on the Haynes lab qPCR machine with melt curve
Results
No template wells did not show any significant fluorescence or peaks on melt curve
KAH154 U20S genomic DNA
finish table
Primer set
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Cp
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Melt curve Tm
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Melt curve area
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Target 1 - primers flanking stop codon insertion site at 72bp |
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Target 2 - primers flanking stop codon insertion site at 162bp |
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|
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Target 3 - primers flanking stop codon insertion site at 255bp |
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|
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GAPDH - primers from KAH |
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|
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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
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
Microscopy on CRISPR'd KAH154 U20S cells
They were all super red.
Flow cytometry on CRISPR'd KAH154 U20S cells
Samples
- No transfection
- Water transfection
- pX330 gRNA 5-1
- pX330 gRNA 5-4
- pX330 gRNA 14-2
- pX330 gRNA 14-3
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.
Put cells on ice, moved to the bench
- 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.
Ran them through the flow cytometer. Didn't analyze the data yet.
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