Biology-Online • View topic - dna ligation protocol for cohesive end
Login

Join for Free!
121850 members
Advertisement
Advertisement

dna ligation protocol for cohesive end

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

dna ligation protocol for cohesive end

Postby genetherapy » Thu Jan 26, 2012 7:46 pm

Hi,
Do anyone suggest me an efficient protocol for cohesive end ligation for cloning?

Thank you
genetherapy
Garter
Garter
 
Posts: 33
Joined: Fri Nov 11, 2011 9:54 am

Postby JackBean » Fri Jan 27, 2012 8:23 am

you mean sticky? What the problem? Just add DNA ligase, some ATP and keep at 17°C or something like that.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Postby genetherapy » Fri Jan 27, 2012 3:54 pm

yes,sticky end ligation. In some protocol it says keep in 16 °C overnight,some says 20°C 4 hours and some says 4°C .I am mixed up :( the ligation ratios of insert and vector also important. In some protocols it says keep the vector and insert at 65°C to avoid selfligation and then put ligase etc. Please help me if there is an effective protocol that works well
genetherapy
Garter
Garter
 
Posts: 33
Joined: Fri Nov 11, 2011 9:54 am


Postby JackBean » Mon Jan 30, 2012 11:09 am

we usually use 16-17°C for O/N. The point of such low temperature is that the sticky ends are short thus the pairing is not much efficient.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 1 guest