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/µL ul for 1ug
KAH77 0.856 0.45 1.903 855.679 1.168662548
KAH78 1.514 0.802 1.888 1513.953 0.660522486
KAH79 0.834 0.439 1.897 833.721 1.19944202
KAH126 0.556 0.294 1.893 556.23 1.79781745
KAH128 0.446 0.235 1.902 446.082 2.241740308
KAH129 0.579 0.305 1.9 579.033 1.727017286

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 reagent 1 25
Optimem 90 2250
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 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


Plate Well DNA PLUS reagent ug DNA Optimem Lipo LTX
1 A1 23 1.82 1 190 3
1 A2 25 1.82 1 190 3
1 A3 29 1.82 1 190 3
1 A4 30 1.82 1 190 3
1 B1 31 1.82 1 190 3
1 B2 32 1.82 1 190 3
1 B3 33 1.82 1 190 3
1 B4 34 1.82 1 190 3
1 C1 35 1.82 1 190 3
1 C2 36 1.82 1 190 3
1 C3 38 1.82 1 190 3
1 C4 39 1.82 1 190 3
2 A1 40 1.82 1 190 3
2 A2 41 1 1 190 3
2 A3 42 1 1 190 3
2 A4 43 1 1 190 3
2 B1 44 1 1 190 3
2 B2 45 1 1 190 3
2 B3 46 1 1 190 3
2 B4 48 1 1 190 3
2 C1 49 1 1 190 3
2 C2 KAH79 1 1 190 3
2 C3 KAH126 1 1 190 3
2 C4 Blank 1 0 190 3