Haynes Lab:Notebook/CRISPR Editing/2015/05/16: Difference between revisions
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
||
Line 13: | Line 13: | ||
:50ng backbone (8486bp) = 50ng /(1000ng/50ul) = 2.5ul | :50ng backbone (8486bp) = 50ng /(1000ng/50ul) = 2.5ul | ||
:10ng insert (824bp) = 10ng / (40ng/25ul) = 6.25ul | :10ng insert (824bp) = 10ng / (40ng/25ul) = 6.25ul | ||
mixed backbone and insert, backbone only, brought both to 10ul with water<br> | |||
added 10ul 2x Roche ligation buffer<br> | |||
added 1ul Roche T4 ligase<br> | |||
mixed and spun<br> | |||
incubated at RT on benchtop for 15-20mins<br> | |||
moved 2ul of each reaction to a new tube, added 10ul water<br> | |||
water only control<br> | |||
added 50ul of my DH5αTs or 50ul of KAH's DH5αTs<br> | |||
incubate on ice for 30min<br> | |||
heat shock at 42°C for 35s<br> | |||
incubate on ice for 2min<br> | |||
add 750ul SOC to each tube<br> | |||
shake at 37°C for 1hour<br> | |||
plate all cells | |||
{| {{table}} | {| {{table}} |
Revision as of 15:11, 16 May 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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
05/16/2015Cloning GFP behind Cas9
mixed backbone and insert, backbone only, brought both to 10ul with water
|