3 replies to this topic

[roelq]

Hi,

I am studying alternative splicing of a certain gene under different conditions.

So I did some PCRs and saw multiple products on gel. I then purified some of these products from the gel and re-did the PCR using the same primers for sequencing. Strangely, I saw that after the 2nd PCR of one of the products (indicated in red on the left gel), I obtained two (maybe even three) products. The size of the smallest product in the middle gel corresponds to the faint band on the left gel. So I purified these two products, again re-did the PCR and still I obtain multiple bands in the reaction with the largest product.

Can anybody explain this?

Cheers,
Roel

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[philman]

non-specific primers? could be that one or both of the primers binds to a second area on the gene, creating a shorter product such as the smaller bands you are seeing!

[roelq]

The primers are really specific.

Anyway, even with non-specific primers you shouldn't get a 200 bp product from a purified 600 bp template.

[aces]

It seems to me that the primers are not specific as you obtain multiple bands from the left gel. And if the primers are non-specific, I couldn't see any reasons why you couldn't get the 200-bp band from the purified 600-bp template.