Thanks for reading. I appreciate your help.
I understand there are some similar topics to this but each is slightly different so I thought I'd post my problem:::
I am trying to insert a 44bp insert into a 7.63KB commercial plasmid.
I am cutting ~ 2.0ug with XbaI and PmeI (Both NEB, in Buffer 4 @37C 1hr)
I then heat inactivate and run on 1% agarose gel, extracting the band with qiagen qiaquick column (horrible yields but much quick than phenol cholorform and no chance of loosing the pellet)
Both enzymes are working, I have run these seperately to check on a gel.
I am not dephosphorylating the plasmid. I get no colonies plating out bacteria transformed with cut plasmid so i think there is no re-ligation happening.
The oligos are IDT standard desalted custom DNA oligos. I am worried about the purification but I know it is working for others too. (I may eventually have to prep a fair few colonies and sequence to check)
I anneal the 2ug of each Oligo according to the protocol, in 50uls annealing buffer
I know that they are annealing (I have checked this on a gel)
My insert is going in according to the commercial protocol which is 50ng plasmid to 4ng insert.
I have tried ligating: (Plasmid: Insert)
All to no avail.
The ligase works (I have tested it each time with singly cut plasmid I am getting about 100 colonies on single digests)
The cells are transforming very efficiently with control plasmids (3000 colonies with 0.1ng Puc1 DNA)
I am getting very low background (1 colony per plate) with no insert so the RE digests are fine.
Because I am doing miRNA target validation I will be trying to do this with a high number of inserts and continously for a fair few months (therefore keeping the costs down is a little important)
If you have any ideas then please let me know.
Thanks so much.
Edited by GilsonGirl, 06 March 2009 - 09:55 AM.