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cloning failure help plzzz

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#1 varuns1982



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Posted 07 July 2011 - 06:53 PM

hi freiends am new in this cloning.. i want to clone my insert of 1.5kb into a transgenic vector having size of 7 kb.
am keep failing by using takara ligation mix.. i tried with 30 min , 1 hr and overnight incubations at 16 degrees. can you suggest some tips for my experiment. i usulally fail to get any colonies and some times i got colonies without insert. help me please

#2 OA17



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Posted 08 July 2011 - 04:54 AM

Hi Varuns1982!

I think it would be better if you gave us more details about your cloning strategy: which restriction enzymes are you using? which is the method that you are using for purifying your bands? Do you run a gel after band purification to check that you are not loosing your product? Does your ligation buffer need ATP? Have you checked the competent cells that you are using? Etc, etc, etc...

Cloning should not be a problem, but there are many steps that should be checked when it doesn't work.

Perhaps if you explain a bit more where you think that there might be a problem, we can give you some advice! ;)

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