Haynes Lab:Notebook/CRISPR Editing/2014/11/23: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2014/11/23 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
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== | ==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. | |||
<br><br> | |||
Set up 4-50μL PCR reactions for untreated Luc14 gDNA and g029 treated Luc14. | |||
Mastermix 1 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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.<br> | |||
PCR <br> | |||
: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 | |||
<br> | |||
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Revision as of 14:44, 24 November 2014
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11/23/2014Thawed Luc14 293 cells, followed Haynes lab protocol
Mastermix 1
Mastermix 1
Transfer 50μL of MM2 into each of 4-PCR tubes. PCR
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