Haynes Lab:Notebook/CRISPR Editing/2015/07/28: Difference between revisions
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'''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?' | ||
* mCherry donor gBlock map: https://benchling.com/s/1dsOuw/edit | |||
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** No template control | ** No template control | ||
* Primer pairs (3 total): | * Primer pairs (3 total): | ||
** P147/148 (flanks stop codons @72) = 148 bp | ** P147/148 (flanks stop codons @72) = 148 bp; Tm ~60°C | ||
** P143/144 (...@162) = 100 bp | ** P143/144 (...@162) = 100 bp; Tm ~55°C | ||
** P145/146 (...@255) = 100 bp | ** P145/146 (...@255) = 100 bp; Tm = 57, 58.8°C | ||
* Number of unique reactions: 8 x 3 = 24 reactions | * Number of unique reactions: 8 x 3 = 24 reactions | ||
* Number of wells needed: 24 x 3 = '''72 wells''' | * Number of wells needed: 24 x 3 = '''72 wells''' (1 plate) | ||
Primer master mixes | |||
* Multiplier = 8 x 3 + 3 = 27 | |||
# P147/148 | |||
# P143/144 | |||
# P145/146 | |||
{| {{table}} | |||
|- | |||
| Reagent || Vol. || (x27) | |||
|- | |||
| 750 nM F/R primer mix || ## || ## | |||
|- | |||
| 2x SYBR (Roche) || | |||
Template master mixes | |||
Revision as of 12:59, 28 July 2015
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07/28/2015
mCherry qPCR/melt pre-course test (at CSH)
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