Problems with Digestion?
Posted 29 May 2013 - 01:57 AM
I am very begginer in this crazy cloning world.
So, I am using PcDNA 3.1 Hygromycin vector (Invitrogen) and I digested them with Fast Digest BamHI and NotI system (Fermentas)
After that I tried the ligation using New England Biolabs T4 polymerase. The buffer, the reactions conditions are all ok.
The problem is that even the controls (digested vector) are growing on LB agar plates.
Do you have any tips or idea where´s wrong?
Thanks in advance
Posted 29 May 2013 - 07:18 AM
Posted 29 May 2013 - 09:06 AM
After running the gel, I purify them. Actually I tried 3 hours digestion with High-Fidelity New England Biolabs BamHI and NotI but it didn´t work as well. I did the dephosphorilation but no results
Is it possible the vector religates during the ligation reaction without the insert? My insert has around 3500 pb
Thanks in advance!
Posted 29 May 2013 - 11:10 AM
Posted 29 May 2013 - 12:41 PM
Posted 29 May 2013 - 08:50 PM
This is what I´ve done. After transformation, plating and overnight culture, I do minipreps and they look very vey crazy...
Thanks in advance,
Posted 09 June 2013 - 11:34 AM