2 bands in same location - (Mar/11/2008 )

Can anyone tell us why this happen.

We exam a gene with alternative splicing with 1 exon deleted.
Our primer cover both side of that exon.
The PCR product is a higher band (300 bp) with that exon in cDNA, and a lower band (170 bp) with that exon deleted.

Our problem now is when we purified band from gel for sequencing, the higher band fragment actually contain both 300 and 170 bp products. Multi templates cause a lot of noise in sequencing result.
Other researcher in our lab even has 4 fragments migrate into same position.

Please can anyone tell us why this happen and how to avoid that in the future.

Thanks

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Not entirely sure why you're getting both bands, unless you're not separating them enough. One thing you could do is subclone the PCR fragments into a vector - like topo ta. Then sequence. You should only get one PCR product inserted into each clone so then you can sequence them cleanly. If you do so, you would probably want to use a polymerase with proofreading activity.

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What sort of gel are you using? If your products are so different in size even agarose electrophoresis should give you really good results. If you are using a polyacrylamide gel that is supposed to separate products of the same size maybe they just happen to have the same denaturing behavour and all you have to do is slightly alter your settings.

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Your most likely problem is a product-dimer of the lower band. It happens frequently on acrylamide. I work on alternative splicing and I get bands I'm not expecting...and more times than not, they turn out to be product dimers.

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The gel I used is 1.5% agarose gel, run at 100V for 60 min.
The 2 bands I am taking about have at leat 1 cm separation on gel.

And for ah6tyfour
Would you kindly let me know how to get ride of product dimer.

My boss think Topo TA cloning too expensive and not necessary to get an ensure result.

Guess this is what we live with

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If you are using agarose, you shouldn't have product dimers. I'm not 100% sure about this though.

Topo TA is around $400, but does include 20 vials of One-Shot Top-10 (and I reguarly use 1/2 vial per experiment).

If you already have competent bacteria lying around (purchased or lab stock), you can just buy the Original TA kit. It's about $180 if you buy it without competent cells. The difference between TOPO TA and Original TA is that you will need to do a normal overnight ligation with the Original TA kit. So instead of doing a 30-minute benchtop ligation and immediately doing the transformation, just set up the ligation before you go home one evening and it will be ready for transformation the next day.

Actually....the best idea may be cloning with a TOPO TA or Original TA kit that you just borrow from the lab next door =) You'd only need enough for one reaction and can just say your PI doesn't want you buying the whole kit to use just once.

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