size problem - (Mar/06/2006 )

Hi, I am trying to ligate a ~5-10 kb part of Candida genome DNA into a pSK+ vector (3kb), but my last 5 ligations failed Could be an insert too big to this vector? I am new to genetics and it amazes me:)
Thanks for answer:)

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This is a loaded question. Are you using whole genome or amplified DNA? Check the compatibility of the insert and vector restriction sites. Used molar ratios that favor the ligation. With a 5-10kb range of fragments you will require different molar rations for the 5kb fragments and 10kb fragments ( and all fragments in between).

Run proper controls for your ligations including a positive control, vector-self ligation and water only to pinpoint any problems.

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try posting the question in the molecular cloning section. the more detail you give, the more we'll be able to help you.

from experience, you have to make an offering to the molecular biology gods before attempting any cloning. they are fickle and probably have psychological problems (you did not hear that from me). but you gotta respect them. i'm not kidding.

vetticus

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vetticus is right. You should really perform the ceremony before planning your experiments. You may even want to hang or post whatever good luck charms you have near and around your thermalcycler (or heat blocks or water baths for ligations).

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