Haynes Lab:Notebook/CRISPR Editing/2015/03/23

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03/23/2015

Cells were about 90-95% confluent so ok to proceed with transfections.
Step 1: Making 1ug/10ul plasmid stocks

Mix plasmids, 0.5ugof each plasmid/transfection, total of 1ug. 8.2x0.5ug DNA
GFP+g034
KAH228+GFP
KAH228+g034

Step 2: make DNA + optimem mixes

GFP+g034 add 320 ul optimem
KAH228+GFP add 320 ul optimem
KAH228+g034 add 320 ul optimem

Step 3: add plus reagent to DNA+ opti

GFP+g034 add 8.2ul plus
KAH228+GFP add 8.2ul plus
KAH228+g034 add 8.2ul plus

step 4: Make optimem + lipo mastermix

Optimem 1233.28
lipo 78.72

Step 5: mix dna mix and lipo mix

GFP+g034 add 410 lipo mix
KAH228+GFP add 410 lipo mix
KAH228+g034 add 410 lipo mix

Step 6: add 100ul dropwise to each well

Nested PCR of Untreated and g025 P178/179 products with P173/215
Nested PCR of Untreated, g044, g048 P178/179 products with P213/214