Blunt-ligating dephosphorylated insert into phosphorylated vectorblunt ligation dephosphorylated i phosphorylated vect
Posted 21 March 2012 - 10:02 AM
For the insert, it's just a PCR product using the Invitrogen High Fidelity Taq Platinum Polymerase (http://tools.invitro...taqhifi_pps.pdf) and dephosphorylated primers.
What ligation kit would you recommend using for this blunt end ligation and what would be the ideal insert:vector ratio to have more plasmids with inserts than auto-ligated plasmids? Also, would it be necessary to purify the PCR product (insert) before ligation? Can someone who has done this help me out? I've done this for awhile, and this is very frustrating for me. Thank you!
Posted 21 March 2012 - 12:00 PM
The optimal ligation ratio is usually reaction dependent. You should set up reactions at 3:1, 6:1 and 10:1 to test this. Note that these are molar ratios not ng amounts!
To calculate the molar ratio use this formula:
ng insert = (ratio) x(length of insert in bp/length of plasmid in bp) x ng plasmid
e.g. if you wanted a 20 ng reaction at a 3:1 ratio with an insert of 300 bp and a plasmid of 3000 you would use ng insert = 3 x(300/3000) x 20...
Posted 21 March 2012 - 02:56 PM
Posted 21 March 2012 - 05:27 PM
You should set up reactions at 3:1, 6:1 and 10:1 to test this. Note that these are molar ratios not ng amounts!
I wonder why many people make this mistake!
Posted 22 March 2012 - 04:06 AM
Well if you dephosphorylate insert you gain nothing. If you don't dephoshorypate vector it will self ligate. So your ligation won't work.
I understand that your method would be the conventional way of solving this problem, but is it possible to ligate the dephosphorylated insert into the phosphorylated vector? Have you attempted or done this before?
It's like using the blacking on the inside of the shoes instead on the outside, serves no purpose.
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Posted 02 August 2012 - 10:22 PM