Haynes Lab:Notebook/CRISPR Editing/2015/09/04

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09/04/2015

Cells in 23 well plates were well spaced, probably about 70% confluent but proceeded with the transfections anyway. Did g025, g031, g044, and g054. Removed media before and added back media without antibiotics.

Step 1: Making .5ug/10ul plasmid stocks ' '
1 well 6.5 wells
ug g034 0.5 3.25
ul g034
ul water
65 total ul
Step 2: make DNA + optimem mixes
1 well 6.5 x
g034 39 add 253.5ul optimem
Step 3: add plus reagent to DNA+ opti
1 well 6.5 x
g034 1 add 6.5ul plus reagent
step 4: Make optimem + lipo mastermix
1 well for 24 wells (x 27)
optimem 47 1269
lipo 3 81
Step 5: mix dna mix and lipo mix
1 well 6.5 x
g034 50 add 325 lipo mix
Step 6: add lipo mix to cells
add 100ul of mix to each well



Removed media from gal4-eed cells that had been under dox for 96 hours. washed with PBS and replaced with media with puro. washed and replaced media on the puro cells as well. will use these for siRNA transfections.