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Problems with Digestion?


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#1 Richard.21

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Posted 29 May 2013 - 01:57 AM

Hello Friends,

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

Johann

#2 Danbas28

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Posted 29 May 2013 - 07:18 AM

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. ..

#3 Richard.21

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Posted 29 May 2013 - 09:06 AM

Hello,

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 Posted Image

Is it possible the vector religates during the ligation reaction without the insert? My insert has around 3500 pb

Thanks in advance!

#4 Danbas28

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Posted 29 May 2013 - 11:10 AM

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.

#5 phage434

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Posted 29 May 2013 - 12:41 PM

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.

#6 Richard.21

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Posted 29 May 2013 - 08:50 PM

Hello,

This is what I´ve done. After transformation, plating and overnight culture, I do minipreps and they look very vey crazy...

Thanks in advance,

#7 phage434

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Posted 30 May 2013 - 05:42 AM

Perhaps you could be more specific than 'crazy'.

#8 notjustalabtech

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Posted 09 June 2013 - 11:34 AM

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).




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