Haynes Lab:Notebook/CRISPR Editing/2014/12/03

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12/03/2014

Need a positive control plasmid for transfections. Measured the concentrations of some candidates from the lab collection:

Sample Read# 260 280 260/280 ng/┬ÁL ul for 1ug
KAH770.8560.451.903855.6791.168662548
KAH781.5140.8021.8881513.9530.660522486
KAH790.8340.4391.897833.7211.19944202
KAH1260.5560.2941.893556.231.79781745
KAH1280.4460.2351.902446.0822.241740308
KAH1290.5790.3051.9579.0331.727017286

Picked KAH79 and KAH126

Transfected each well of two 12-well plates with 1ug of plasmid DNA.

Step 1: Make mastermixes

MM1: Mix PLUS reagent with optimem

' 1 rxn 25 rxns
PLUS reagent125
Optimem902250
I messed this up, it's reflected in the table below showing that some wells had 1.82ul of PLUS reagent.

MM 2: Lipo LTX and optimem

' 1 rxn 25 rxns
Lipofectamine LTX375
Optimem1002500


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


Plate Well DNA PLUS reagent ug DNA Optimem Lipo LTX
1A1231.8211903
1A2251.8211903
1A3291.8211903
1A4301.8211903
1B1311.8211903
1B2321.8211903
1B3331.8211903
1B4341.8211903
1C1351.8211903
1C2361.8211903
1C3381.8211903
1C4391.8211903
2A1401.8211903
2A241111903
2A342111903
2A443111903
2B144111903
2B245111903
2B346111903
2B448111903
2C149111903
2C2KAH79111903
2C3KAH126111903
2C4Blank101903


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