TOPO TA cloning problem - (Apr/02/2010 )

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molstudent on Apr 10 2010, 04:11 AM said:

I tried the new primers and still same result. Only getting 1 or 2 white colonies per plate and they are negatives. I cant figure out why my PCR product isn't getting into the TOPO vector. Since i run it on a gel and purify it i know my fragment is the correct size so the PCR worked. what is causing this?

Did this "1 or 2" colonies in your plate contain your "vector with pcr insert"?

-adrian kohsf-

What is your insert:vector ratio in your ligation reaction? In some stubborn clonings, I have gone up to 6ul insert, and keeping the salt and vector the same as recommended.

Also, how long are you leaving the ligation at RT? Contrary to what the invitrogen manual says, I sometimes find that longer than 5 minutes is required to get my insert. I have even left a ligation at RT for up to 4 hours and had plenty of clones to work with. This same cloning that worked with 4 hour ligation failed twice with the recommended 5 minutes.

-BryanC-

the 1 or 2 colonies sometimes have my insert and vector and sometimes they dont but more often they dont.

i am ligating for 30 minutes and im not sure of the ratio. i just use 1ul of salts and have tried .5ul and 1ul of vector and i fill the rest with pcr product (gel purified). i have also tried 1ul of vector and 1ul of insert and the rest water, with no salts. i get the same outcome for each method.

-molstudent-

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