11/23/2014
Thawed Luc14 293 cells, followed Haynes lab protocol
- Transfer 5 mL of pre-warmed 100% FBS into a 15 mL conical tube.
- Retrieve the frozen cell vial from the -150°C freezer.
- Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed.
- Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS.
- Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes.
- 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.
- Flick the tube to gently resupend the pellet in the ~100 uL FBS. Had trouble with this, may have been too rough.
- Stand the t-25 flask up and remove the cap.
- Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
- Transfer the cells + medium into the T-25 flask.
- 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.
- 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
|
Project name
Main project page
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