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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png 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>      </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>


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

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