Haynes Lab:Notebook/CRISPR Editing/2015/01/25

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01/25/2015

Picked KAH79 and KAH126

Transfected each well of two 12-well plates with 1ug of plasmid DNA. The cells looked way better than last time. Luc14s were about 80-90% confluent while the gal4-eeds were about 90-95% confluent. The media for the gal4s was starting to turn orange so I added 1mL of media to each well (added dox to the gal4s) Step 0: Prep DNA
definitely g023, g034 for the third: in order of importance: g048, g046, g044, g025 need 3 tubes with 1ug/10ul each.
controls:

1.8ul KAH126 + 8.2ul elution solution
10ul elution sol'n


Step 1: Make mastermixes

MM1: Mix PLUS reagent with optimem

' 1 rxn 25 rxns
PLUS reagent 1 25
Optimem 90 2250

MM 2: Lipo LTX and optimem

' 1 rxn 25 rxns
Lipofectamine LTX 3 75
Optimem 100 2500


Step 2: Mix MM1 with DNA

Add 91ul of MM1 to 1ug DNA in 10ul of Elution solution
Incubate for 5 minutes at RT

Step 3: Mix in Lipo LTX

Add 100ul of MM2 to DNA-MM1
Incubate for 30 minutes at RT

Steo 4: Add complexes dropwise to each well
Everything went well, finished about 4:30.



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