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'''
'''QPCR of genomic DNA isolated from KAH154 U20S cells'''
<br><br>
<br><br>
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| Rxn 8||no template||GAPDH - primers from KAH||3
| Rxn 8||no template||GAPDH - primers from KAH||3
|}
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''Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.''
<br><br>
<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]]
[[Image: 14.07.19 qPCR plate layout.png| 500px | plate layout]]


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#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
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#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.


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Revision as of 21:27, 23 July 2014

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07/19/2014

QPCR of genomic DNA isolated from KAH154 U20S cells

' Template DNA Gene Target (primers) 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.

plate layout

Gene Target (primer) master mixes

Gene Targets: 4 tubes

  1. Target 1 - primers flanking stop codon insertion site at 72bp
  2. Target 2 - primers flanking stop codon insertion site at 162bp
  3. Target 3 - primers flanking stop codon insertion site at 255bp
  4. 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

  1. untreated KAH154 U20S cells
  2. no template


10ng of genomic DNA used for each qPCR reaction
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

  1. Pipette 13.5 µL of the appropriate Template master mix into the first well of each 3-well group.
  2. 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.
  3. 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.
  4. 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 Cp Melt curve Tm 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


Conclusions

  1. Order new primers for Target 1
  2. Targets 2 and 3 work well
  3. 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

  1. No transfection
  2. Water transfection
  3. pX330 gRNA 5-1
  4. pX330 gRNA 5-4
  5. pX330 gRNA 14-2
  6. pX330 gRNA 14-3


Harvesting the cells

  1. Removed media
  2. Washed with PBS
  3. Added 0.5mL of trypsin to each well
  4. Incubated at RT for 8 minutes
  5. Added 1.0mL media
  6. Loosened attached cells by pipetting. Got most of the cells up
  7. Collect the cells in growth medium and transfer to 15 mL conical tubes.
  8. Pellet the cells at room temperature at 1000 rpm for 3 min.
  9. 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.
  10. 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

  1. 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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