Haynes Lab:Notebook/CRISPR Editing/2015/01/25: Difference between revisions
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==01//2015== | ==01/25/2015== | ||
Picked KAH79 and KAH126<br><br> | |||
'''Transfected each well of two 12-well plates with 1ug of plasmid DNA.''' | |||
The cells looked way better than last time. Luc14s were about 80-90% confluent while the gal4-eeds were about 90-95% confluent. The media for the gal4s was starting to turn orange so I added 1mL of media to each well (added dox to the gal4s) | |||
'''Step 0: Prep DNA'''<br> | |||
definitely g023, g034 | |||
for the third: in order of importance: g048, g046, g044, g025 | |||
need 3 tubes with 1ug/10ul each. | |||
<br> | |||
controls: | |||
:1.8ul KAH126 + 8.2ul elution solution | |||
:10ul elution sol'n | |||
<br> | |||
'''Step 1: Make mastermixes'''<br> | |||
MM1: Mix PLUS reagent with optimem | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''25 rxns''' | |||
|- | |||
| PLUS reagent||1||25 | |||
|- | |||
| Optimem||90||2250 | |||
|} | |||
MM 2: Lipo LTX and optimem | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''25 rxns''' | |||
|- | |||
| Lipofectamine LTX||3||75 | |||
|- | |||
| Optimem||100||2500 | |||
|} | |||
<br> | |||
'''Step 2: Mix MM1 with DNA''' | |||
:Add 91ul of MM1 to 1ug DNA in 10ul of Elution solution | |||
:Incubate for 5 minutes at RT | |||
'''Step 3: Mix in Lipo LTX''' | |||
:Add 100ul of MM2 to DNA-MM1 | |||
:Incubate for 30 minutes at RT | |||
'''Steo 4: Add complexes dropwise to each well''' | |||
<br> | |||
Everything went well, finished about 4:30. | |||
Revision as of 16:33, 25 January 2015
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||
01/25/2015Picked KAH79 and KAH126
MM1: Mix PLUS reagent with optimem
MM 2: Lipo LTX and optimem
Step 3: Mix in Lipo LTX
Steo 4: Add complexes dropwise to each well
|