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
|
| 
Project name
