Haynes Lab:Notebook/CRISPR Editing/2016/02/23: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 57: Line 57:
:1ul Cutsmart buffer or Fastdigest
:1ul Cutsmart buffer or Fastdigest
2ul plasmid, 10ul total rxn<br>
2ul plasmid, 10ul total rxn<br>
Incubate at 37 for 1 hour<br>Confirm on gel
Incubate at 37 for 1 hour<br>Confirm on gel<br>
SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.<br>XbaI cuts in cutsmart, SgrAI cuts in FD buffer.
<br><br>
<br><br>


Revision as of 11:47, 27 February 2016

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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

02/23/2016

Test qPCR primers for ChIP
Dilute Gal4-EED 1 sonicated and column purified DNA from ChIP 1:100
Each reaction

1ul diluted template
1ul F primer
1ul R primer
7ul water
10ul Taq 2x MM


Tm 57

Tube F primer R primer amplicon length Tm for taq
1 P313 P311 186 54
2 P309 P160 171 54
3 P309 P312 175 56
4 P308 P160 202 54
5 P308 P311 171 54
6 P314 P318 205 47
7 P316 P317 169 48
8 P315 P318 197 47
9 P316 P318 160 47
10 P316 P214 196 48



Cut MV13-KAH60
Plasmid: MV13-KAH60 6 297ng/ul, confirmed by Cass
Test SgrAI

1ul SgrAI
1ul Fastdigest buffer or Cutsmart


Test XbaI

1ul XbaI
1ul Cutsmart buffer or Fastdigest

2ul plasmid, 10ul total rxn
Incubate at 37 for 1 hour
Confirm on gel
SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.
XbaI cuts in cutsmart, SgrAI cuts in FD buffer.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/CRISPR_Editing/2016/02/23&oldid=932245"