Haynes Lab:Notebook/CRISPR Editing/2015/07/28: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 15: Line 15:
'''mCherry qPCR/melt pre-course test (at CSH)'''
'''mCherry qPCR/melt pre-course test (at CSH)'''
* The situation: In 2014, a single gBlock was created as the template to generate mutational donor sequences for CRISPR/HDR at different positions within mCherry in KAH154 cells. Double stops were added at positions 72, 162, and 255. The plan was to PCR-amplify three different donors to insert stops at each position in separate experiments. Because of a primer shipping delay, we could not generate these separate donors as planned, and instead used the whole gBlock as the donor. So, we now have an accidental experiment where the question is 'did all three stop-mutations get inserted in a single CRISPR treatment?'
* The situation: In 2014, a single gBlock was created as the template to generate mutational donor sequences for CRISPR/HDR at different positions within mCherry in KAH154 cells. Double stops were added at positions 72, 162, and 255. The plan was to PCR-amplify three different donors to insert stops at each position in separate experiments. Because of a primer shipping delay, we could not generate these separate donors as planned, and instead used the whole gBlock as the donor. So, we now have an accidental experiment where the question is 'did all three stop-mutations get inserted in a single CRISPR treatment?'
Calculate number of wells
* Templates (8 total):  
* Templates (8 total):  
** From 2014: gDNA U-2 OS KAH154: CRISPR 1d, 2d, 4d
** From 2014: gDNA U-2 OS KAH154: CRISPR 1d, 2d, 4d

Revision as of 12:44, 28 July 2015

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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

07/28/2015

  • mCherry qPCR/melt pre-course test (at CSH)


mCherry qPCR/melt pre-course test (at CSH)

  • The situation: In 2014, a single gBlock was created as the template to generate mutational donor sequences for CRISPR/HDR at different positions within mCherry in KAH154 cells. Double stops were added at positions 72, 162, and 255. The plan was to PCR-amplify three different donors to insert stops at each position in separate experiments. Because of a primer shipping delay, we could not generate these separate donors as planned, and instead used the whole gBlock as the donor. So, we now have an accidental experiment where the question is 'did all three stop-mutations get inserted in a single CRISPR treatment?'

Calculate number of wells

  • Templates (8 total):
    • From 2014: gDNA U-2 OS KAH154: CRISPR 1d, 2d, 4d
    • From 2015 (new): 3d (mixed population...transfected w/ p330-3T or pX330-3B + PCR donor Stop@162)
    • From 2015 (new): PCR'ed dsDNA donor Stop@72, Stop@162, Stop@255
    • No template control
  • Primer pairs (3 total):
    • P147/148 (flanks stop codons @72) = 148 bp
    • P143/144 (...@162) = 100 bp
    • P145/146 (...@255) = 100 bp
  • Number of wells needed: 8 x 3 x 3 = 72 wells




Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/CRISPR_Editing/2015/07/28&oldid=895058"