User:Karmella Haynes/Notebook/PcTF Genomics/2014/09/09

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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):

  1. KAH160/015 (8/09/10)
  2. KAH160/015 (9/07/14) --> new miniprep
  3. KAH165/015 (8/09/10)
  4. KAH165/015 (9/07/14) --> new miniprep
  5. KAH170/015 (8/09/10)
  6. KAH170/015 (9/07/14) --> new miniprep
  7. 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
KAH160/015 8/09/10 0.155 1.88 154.5 2163.0/ 14 μL
KAH160/015 8/09/10 1.210 1.89 210.2 2522.4/ 12 μL + 2 μL H2O


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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