Haynes Lab:Notebook/CRISPR Editing/2015/01/25: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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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.




Latest revision as of 00:40, 27 September 2017

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01/25/2015

Picked KAH79 and KAH126

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
definitely g023, g034 for the third: in order of importance: g048, g046, g044, g025 need 3 tubes with 1ug/10ul each.
controls:

1.8ul KAH126 + 8.2ul elution solution
10ul elution sol'n


Step 1: Make mastermixes

MM1: Mix PLUS reagent with optimem

' 1 rxn 25 rxns
PLUS reagent 1 25
Optimem 90 2250

MM 2: Lipo LTX and optimem

' 1 rxn 25 rxns
Lipofectamine LTX 3 75
Optimem 100 2500


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
Everything went well, finished about 4:30.



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