I really need some advice on ligation. This is the thing, I've managed to PCR a 300 bp product, using a forward primer (with an EcoRI site) and a reverse primer (BamHI site). I'm purifying the product with QIAgen, double digesting it with EcoRI and BamHI...meanwhile, double digesting pBluescript KS+ also with EcoRI and BamHI....treating the cut pBSKS+ with CIAP... gel purifying it.... ligating the double digested 300 bp product into the cut pBSKS+ (about 50ng) in a 3:1 molar ratio of insert: vector, using NEB T4 ligase and buffer, at room temperature (usually overnight, but at least 2 hours)... ethanol precipitating it, transforming it onto electrocompetent cells, and plating it O'night... blue white selection...controls of vector and ligase, and vector with no ligase... anyway, i'm lucky to get a positive colony on my test plates... and with my one and only lovely positive colony, which I had pinned all my hopes on , minipreped...double digested again using EcoRI and BamHI...ran an AGE gel to see if there was insert...and there was none, just bands at around 3000bp and higher. (ok, occasionally i've gotten the insert, and then its party time...but really, it's getting ridiculous,)
I've repeated this again, and again.... I've used from 20 to 100 ng of vector, I've used 2:1, 3:1, 4:1, 100:1 ratios. I even whispered sweet nothings into its ear.
WHY IS THIS GOING SO WRONG?? I was thinking that the pBluescript is ligating to itself to make a "super big huge plasmid"... getting into the cells and appearing white. But, to the point, why isn't the insert being shoved into the stinking little vector, and doing what it's supposed to, and why isn't this super huge big plasmid being shown on the gel when i was looking at how much background to expect?? with the ligations that i've run on the gel, it looks like there is no, and I mean no, background, it's lovely and clean, nice band of cut vector, nice band of insert... absolutely nada in the region where the vector + insert in a circle should be.
I've had it up to here with this, first the PCR wouldn't work for over a month because the bloody polymerase was dead/dying, the primers that were suggested to be used were stuffed, and the temperatures that were used were completely out of whack...finally get that to work, happy time , cloning this should be a cakewalk, but it's not. WHY ME??
can anyone help me out?
I was thinking that since my PCR used primers with the EcoRI and BamHI site, maybe they don't need to be digested? Maybe the ligation should be in a 37' water bath, or in the fridge, or spinning, or far, far away from me. HELP!
I really don't know what to do. I will listen to *ANY* suggestions.
Edited by vetticus3, 28 September 2004 - 06:27 PM.