Haynes Lab:Notebook/CRISPR Editing/2014/11/23

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11/23/2014

Thawed Luc14 293 cells, followed Haynes lab protocol

  1. Transfer 5 mL of pre-warmed 100% FBS into a 15 mL conical tube.
  2. Retrieve the frozen cell vial from the -150°C freezer.
  3. Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed.
  4. Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS.
  5. Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes.
  6. After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet.
  7. Flick the tube to gently resupend the pellet in the ~100 uL FBS. Had trouble with this, may have been too rough.
  8. Stand the t-25 flask up and remove the cap.
  9. Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
  10. Transfer the cells + medium into the T-25 flask.
  11. Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
  12. Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight.



Set up 4-50μL PCR reactions for untreated Luc14 gDNA and g029 treated Luc14.

Mastermix 1

' 1rxn 8.3 xns
H20 30 249
HF 5X MasterMix 10 83
dNTPs 1 8.3
FP 2.5 20.75
RP 2.5 20.75
46 381.8

Mastermix 1

' 1rxn 4.1 xns
MM1 46 188.6
Template 2 8.2
DMSO 1.5 6.15
Phusion 0.5 2.05
50 205

Transfer 50μL of MM2 into each of 4-PCR tubes.

PCR

98°C for 3 min
36 cycles
98°C for 10s
66°C for 30s
72°C for 60s
72°C for 10 min
4°C forever