Blunt-ligating dephosphorylated insert into phosphorylated vector - (Mar/21/2012 )
I have been currently trying to blunt ligate a dephosphorylated insert (~3.4kb) into a phosphorylated vector (~8.0 kb), but so far this hasn't worked. I've linearized the phosphorylated vector using the HindIII enzyme and then inactivated it in high temperature. After, I've blunted it using the mung bean nuclease, and then gel-extracted it to get rid of the non-linearized plasmids.
For the insert, it's just a PCR product using the Invitrogen High Fidelity Taq Platinum Polymerase (http://tools.invitrogen.com/content/sfs/manuals/platinumtaqhifi_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!
Your problem probably lies in that you should de-phosphorylate your plasmid so as to prevent self-ligation, seeing as you only cut with one enzyme which can result in re-ligation.
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...
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?
bob1 on Wed Mar 21 20:00:33 2012 said:
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!
hksup2001 on Wed Mar 21 22:56:00 2012 said:
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?
Well if you dephosphorylate insert you gain nothing. If you don't dephoshorypate vector it will self ligate. So your ligation won't work.
It's like using the blacking on the inside of the shoes instead on the outside, serves no purpose.
You can phosphorylate the insert using T4 polynucleotide kinase - note that this is different to the T4 ligase!
people do phosphorylate vector and use dephosphorylated primers and nicely clone it. But you need activation of suicide gene in vector if you vector is trying to self ligate. Read about PJET 1.2 vector cloning strategy basics.