I am new to this site and I hope to get some help from you guys.
I have been trying to construct a vector for months. I am currently facing some weird problems that I cannot solve or explain!!
So what I am aiming to do is to clone a 3kb DNA into a vector and then insert a loxp site into the vector construct.
Things that I have done:
1. PCR the DNA and inserted it into the vector
2. sequencing result is good
then is the weird thing:
I cut the vector+insert with a RE (BlpI) and try to clone the loxp in.
colonies are grown on Amp resistant agrose plate.
colonies ratios of ligation plate and control plate is ~8:1 (not bad).
then I did miniprep with Qiaprep spin column to isolate DNA
I did double digestion to check the loxp site. The two restriction sites are on each side of the loxp (~400bp)
no DNA from miniprep can be cut!!! ( but I can see DNA on the gel as a clear band, which is about the right size of vector+insert).
I ran a control of test digestion with untreated vector+insert (the sample that I sent for sequencing before). the double digestion worked well.
I sent a few for sequencing, the results are all negative.
I also tried to PCR the loxp region (~300bp). It only worked for the untreated vector+insert, but not the DNA from miniprep (no amplification at all).
I repeat this twice and it all give me the same result.
I wonder if those are just garbage DNA, where they came from and how to solve the problem
Plz help, I am really frustrating and depressed.
Edited by yesandno, 09 August 2009 - 08:20 PM.