I'm trying to clone a PCR product of 750 bp amplified from DNA extracted from environmental samples. I used a "Wizard PCR and Gel clean-up system" to purify the gel band of my product (I'm always careful to expose the gel to UV less time as possible). For transformation i'm using chemocompetent DH5-alpha, with a heat-shock of 42°C at 45 seconds.
I'm using pGEM or pJET as vector, but I get less than 40 transformant colonies (but I need 100+ for clone library). In my transformation control I get always 100+ colonies ( I use 1 ul of miniprep purified pGEM with an insert) ; but when I use a ligation control for pJET, I also get 40 or so transformants.
I've previously done the ligation and transformation using pJET and DH5a with a fragment of 370 bp also amplified from environmental DNA; obtaining 647 transformants with my insert.
I suspect there is a problem with ligation, based on the controls. Is there an upper limit of insert DNA (purified or direct from PCR) quantity during the ligation? Should I increase the time of ligation?
I've repeated the PCR + ligation + transformation changing different parameters, but I still get less than 40 (white when i use pGem) colonies. I really need some advice of what to do or what may I have forgotten.
Edited by Procyon, 31 January 2013 - 07:59 AM.