Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30

From OpenWetWare
Jump to navigationJump to search
Ligation/Amplification of DBN002 & DBN003 <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>

Overview

Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification.

Type IIS Digestion/Ligation

Reagent Volume (µL)
Insert 1 (DBN002) 200 ng (11.9 µL)
Insert 2 (DBN003) 500 ng (7.5 µL)
10x FD Buffer (clear) 2.5
10mM DTT 1
10mM ATP 1
FD BsaI 1
T4 DNA ligase 0.5
M.B. H2O 0
Total 25.4

Incubate the ligation reaction in a thermocycler (program name DBN IIS):

  1. 37°C 5 min
  2. 23°C 5 min
  3. Cycle the previous two steps for 6 cycles (total run time 1h)
  4. 4°C hold until ready to proceed

Clean-up of IIS Ligation

Use the Qiagen kit. If possible, final volume elution of 30 µL.

PCR

Reagent Volume (uL)
2x master mix 25
MB grade H2O 0
DBN003 f1 primer 0.25
DBN002 r1 primer 0.25
template 24.5
Total 50
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x40):
denature 95 30 sec
anneal 59 30 sec
extension 72 120 sec
final extension 72 300 sec
soak 4 indef.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR/2015/04/30&oldid=882946"