Simple sublconing not working - why? (Oct/24/2007 )
QUOTE (newboy @ Nov 1 2007, 08:26 PM)
I got the similar trouble.
I did the same process to subclone my insert to a expression vector. I did several subclone before with the same process. but it won't work in this new lab. I do not know where is the point. I add 3 extra bp to the outside of the enzyme site for digestion according NEB website suggestion (it works for my previous work). But I cannot get the result. I suspect many things but I cannot change all the staff for the single construct as I am the new guy here although no one else works on this cloning staff now.
I called NEB. They suggestion add 6 bp for sufficent digestion. Anyone have suggestion for the 6bp. what should I take? GCTACG?
After transformaion, I cannot get any colonies on the plate. So I just worry about my ligase and competent cells instead of my digestion.
Question, can I use qiagen gel extract kit or PCR extract kit to remove digestion staff and enzyme instead of run a gel? which kit works better?
I did the same process to subclone my insert to a expression vector. I did several subclone before with the same process. but it won't work in this new lab. I do not know where is the point. I add 3 extra bp to the outside of the enzyme site for digestion according NEB website suggestion (it works for my previous work). But I cannot get the result. I suspect many things but I cannot change all the staff for the single construct as I am the new guy here although no one else works on this cloning staff now.
I called NEB. They suggestion add 6 bp for sufficent digestion. Anyone have suggestion for the 6bp. what should I take? GCTACG?
After transformaion, I cannot get any colonies on the plate. So I just worry about my ligase and competent cells instead of my digestion.
Question, can I use qiagen gel extract kit or PCR extract kit to remove digestion staff and enzyme instead of run a gel? which kit works better?
Some enzymes require 6 bases for efficient digestion like Not1. We usually add 6 A's or T's at the end of the primer for proper digestion.
Some people run the PCR on gel and purify and others use PCR purification kit. We run it on the gel and purify it before digesting with the enzymes.
-scolix-