No insert in subcloning - (Nov/10/2012 )
I've cloned a 1700 PCR product into a pJet vector - went perfectly allright. I want to transfer the insert into a pCMV5. The insert itself have EcoRI at the beging and BglII restriction site at the end. I double digest the pJet-Insert plasmid and ive got perfect bands on the gel. I isolate the band corresponding to the insert and clean it up with Axygen gel extraction kit. I double digest the pCMV5 vector and dephosphorylate it with TSAP, then clean it up with axygen pcr cleanup kit. I take 50 ng of digested and dephosphorylated vector and 3,4,5 times more (molar) of the digested insert, then ligate it with NEB quick ligase, directly transform the NEB DH5a competent cells ( full protocol) and spread them on LB-amp agar. Overnight i always get around 20 colonies per plate. After an hour outgrowth in LB i make a colony pcr for the insert and always analyse ALL the colonies (around 60 from all the plates)! Ive tried it 4 times now, and ive got NO insert whatsoever!! WHAT AM I DOING WRONG couse i'll go insane in the moment!
How do your control plates look? It sounds like you have some backgound un-digested vector.
Is the ligase and ligase buffer fresh? - these have/require ATP in them, which degrades quickly with freeze/thaw. You can supplement the buffer with ATP if necessary, but it is best to aliquot the tube and only freeze/thaw once.
Check that both your REs are working properly.
Edited to add: you don't need to de-phos, BglII and EcoR1 are not compatible ends, so you should only have minimal self-religation.
I actually do the exact opposite with inserts and vectors. If I'm only getting one good strong band from PCR, I usually just digest the insert, then PCR cleanup only to keep away from the contaminants from a gel extraction (I only gel extract if I'm getting multiple bands). But I always gel extract the cut vector, since you want to avoid getting uncut, or supercoiled DNA in with your ligation at all costs (you don't want single-cut either, but that's usually hard to spot on a gel). The contaminants I normally get from gel extraction are bad enough that I've re-done my PCR off my gel extracted insert, then run a PCR cleanup off that to keep the insert I use in the ligation as clean as possible.
As bob1 said, you almost surely just have uncut vector contaminating your ligation. Without a control vector-alone ligation, you have no way of knowing how many colonies is reasonable to screen for insert. If you don't see more than 2:1 colonies on your +insert plates versus your control plates, I'd consider it a failed ligation and just repeat the cloning from the "last known good" step (or even from the beginning if I'm feeling paranoid). If you're only at 1.5:1, then you could probably try 10 colonies or something to try to salvage the work you've done, but I'd simultaneously restart the cloning so I can hit the ground running if none of them have insert.
it all comes down to the fact that i'm an idiot:) i had restrictases sites one next to the other in the MCS, and BglII needs at least 3bp overhang to work( after EcoRI digestion there was only 1bp overhang). So as the EcoRI is a hyperactive cyborg comparing to BglII, my vector was just single cut. Sequential digeston, first BglII, then EcorI made it work!! Always look at the vector map!!