Problems with Digestion? - (May/29/2013 )
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
Did you elute your DNA directly from digestion or after gel running? in the second way it would be more specific. They're both sticky ends, it has not to be difficult to re-ligate . You also can try to digest DNA 1-2 hours more, it will be more efficient. ..
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!
Yes, it's possible, even if the phosphatase treatment minimize self-ligated vector. One thing that you could do is a PCR screening of some colonies, or definitely you could verify the plasmid heights from a gel after some minipreps of some colonies.
Background can be reduced substantially by amplifying the vector backbone with primers containing your desired restriction sites. Template can be removed by digestion with DpnI following PCR. If you use very small amounts of template, there is little circular DNA left wihch can transform.
This is what I´ve done. After transformation, plating and overnight culture, I do minipreps and they look very vey crazy...
Thanks in advance,
Perhaps you could be more specific than 'crazy'.
Yes, it is always possible that your plasmid could religate without the insert unless you get rid of it using gel extraction or Dpn1 digestion after amplification. You could ligate overnight (16C).