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problem with pLK01 plasmid ligation


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#1 sick of cloning

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Posted 12 October 2010 - 03:14 PM

Hi,
I am trying to ligate a 4.9kb insert into pLK01 plasmid which is 8.9Kb using NruI/EcoRI restriction sites.When I cut the plasmid with these two enzymes I get two pieces: 3.3Kb and 5.6Kb.However after ligation of 4.9Kb insert,when I cut using the same enzymes I get different sizes from the plasmid:4.3Kb and 6.5Kb which are not compatible with my insert and backbone sizes.It seems like the insert size decrease and the backbone size increase!!!Has anyone seen something like this before?or what could be causing it?

I have been trying to clone this for 3 months and it is really frustrating!
Please help!!!any suggestion is appreciated!!!

Sick of Cloning

#2 phage434

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Posted 12 October 2010 - 05:33 PM

So, have you tried figuring out what your mystery plasmid is? Have you digested with other restriction enzymes? Have you sequenced it? Figuring out what is wrong is often the first step in fixing it. Trying it again blindly won't get you anywhere.

#3 sick of cloning

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Posted 13 October 2010 - 06:05 AM

Yes I tried digestion with other enzymes.
1-I tried SmaI which is unique for my insert,the plasmid become linearized which means insert exist there but when I use other enzymes such az NruI/EcoRI to check the sizes,the sizes are incorrect.
2-I tried BamHI which is in both insert and vector,it just become linearized.
3-I tried NcoI which is in both insert and vector,the sizes it gives me is not correct.
I did not sequence it,but it is a good suggestion,Thanks.




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