Very, VERY weird PCR results! - (Feb/08/2006 )

I have been strugling with cloning of a really big gene (size 2.1kb) When I get my transformants, I get really weird results with PCR: I try to look for the fragment with different internal primers and I do get the desired expected band with every primer pair, but when I run a sequencing reaction, my insert isn't there. How weird is that!?

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It could be contamination. Have you run negative controls i.e. PCR reaction without DNA?

You could also try to identify the insert by digestion, if it's applicable.

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Negative controls are not giving me anything, so it's not contamination. Digestion is what I am doing right now, but it'ss till weird that a positive PCR with 2 internal primer pairs are not a guarantee I have an insert.

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Hiii...
It so happens some time that primers give non specific amplification of the same size... when got sequenced it turns out to be alien... i suggest you best to check the insert is by digestion and then get the pop out sequenced.. that will be best.

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with an insert that size you should be able to see a difference in size between a positive clone and your plasmid alone. what do they look like on a gel? if the suspected clone is heavier then you best look at your sequencing rxn.

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