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09/09/14
- Lenti Transfection: Day 1 - lipo transfect HEK293 w/ FUW plasmids (LaBaer lab)
- Transformation: pLenti4 plasmid (alternative backbone to FUW/KAH015)
Lenti Transfections
- Used 6-well plate Lipofectamine protocol
- Requires ~2000 ng in 20 μL vol. per 600 μL Lipo complex mix
- LaBaer lab protocol: Required DNA for virus (mass ratio = 5:5:1)...
- ORF-carrying plasmid (5)--> target = 2500 ng in 14 μL
- dR8.91 packaging plasmid (5) --> 5 μL (500 ng/μL, from LaBaer lab)
- VSVG packaging plasmid (1) --> 1 μL (500 ng/μL, from LaBaer lab)
- The total mass for this is 5500 ng; decided to mix this amount in 20 and then use half (2250 ng) for Lipo + 10 μL water (stored other half as back-up)
My ORF-carrying plasmids (& 6-well plate labels):
- KAH160/015 (8/09/10)
- KAH160/015 (9/07/14) --> new miniprep
- KAH165/015 (8/09/10)
- KAH165/015 (9/07/14) --> new miniprep
- KAH170/015 (8/09/10)
- KAH170/015 (9/07/14) --> new miniprep
- Turbo GFP control, 980 ng/μL, used 2.5 μL (LaBaer lab)
- New minipreps had low concentrations (~20 ng/μL, 100 μL total)
- Zymo-cleaned total minipreps (9/07/14 samples) to get ~2000 ng in 14 μL H2O
- Also measured conc. of old plasmids (8/09/10) - was in a hurry, so no time to clean and concentrate old plasmids
| Sample |
OD260 |
260/280 |
ng/μLOD260 |
ng/vol for Transf.
|
| KAH160/015 8/09/10 |
0.086 |
1.87 |
86.2 |
1206.8/ 14 μL
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| KAH160/015 8/09/10 |
0.155 |
1.88 |
154.5 |
2163.0/ 14 μL
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| KAH160/015 8/09/10 |
1.210 |
1.89 |
210.2 |
2522.4/ 12 μL + 2 μL H2O
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Lipo complexes (done in LaBaer TC room)
- 10 μL of ORF/dR8.91/VSVG mix
- + 10 μL DEPC water
- + 570 μL OptiMEM (Haynes Lab)
- + 2.5 μL PLUS reagent (Haynes Lab) --> 5 min. room temp
- + 7.5 μL Lipo LTX (Haynes Lab) --> 30 min. room temp
Transfections
- HEK293 grown in 2 mL DMEM/FBS (no pen-strep)
- Removed 1 mL old medium
- Carefully & slowly added 1 mL fresh medium (cells easily detach if not careful)
- Added Lipo/DNA complexes drop-wise
- Spun plates for 30 min (Seron's protocol - 'helps complexes to enter cells')
- (Seron) placed cells into 37°C incubator
Note: return to lab at 10 am tomorrow to change out medium
Transformation - pLenti4 plasmid
- Dilute 0.5 μL DNA in 10 μL dH2O
- Add to 50 μL DH5α-turbo
- Incubate on ice/ 5 min.
- Spread onto 100 μg/mL Amp plates
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Pc-TF Genomics
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