Double bands in GAPDH

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Debiparna
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Double bands in GAPDH

I am getting double bands in RT PCR of my GAPDH..have tried a lot of ways..no use...could u suggest something?

itaylor
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If I'm not mistaken. Gapdh

If I'm not mistaken. Gapdh has several pseudogenes depending on species. So the artifact you are seeing may be real.

Firoz Ahmad
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Debiparna wrote:I am getting

Debiparna wrote:

I am getting double bands in RT PCR of my GAPDH..have tried a lot of ways..no use...could u suggest something?

Hi!
I am using GAPDH as an internal control but i never faced such typle of problems! well probably by changing the annealting temp or reducing the no of cycle can short out yr problem!
Thanks

Tony Rook
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Debiparna:

Debiparna:

Are you performing Real Time PCR or end point? If you are using real time, maybe you could confirm the two amplicons with a SYBR Green melting curve.

Marina Fomin
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trook wrote:Debiparna:

trook wrote:

Debiparna:

Are you performing Real Time PCR or end point? If you are using real time, maybe you could confirm the two amplicons with a SYBR Green melting curve.

I have this problem: one really sharp pick on melting curve but two or more bands on gel. Do you have any suggestion?

Tony Rook
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Marin Fomin:

Marin Fomin:

You're issue may be due to one of two issues (as I see it). You may have two amplicons with exactly the same melting point. This is not entirely impossible.

Or, one of you're bands may be melting below where you have begun collecting data for your melting curve.

Depending on how important it is to keep you're designed primer set. You can either design a new set of primers or confirm you have only one amplicon through using TOPO TA kit (available through Invitrogen - these kits might be expensive) to perform a ligation to add several more nt to your amplicon. The appearance of two melting curves following this would confirm you're band data.

Good luck and please let the board know how it turns out.

Tony Rook
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Marin Foman:

Marin Foman:

Here is a link for the User's Manual for the TOPA TA Five-minute cloning of Taq polymerase-amplified PCR products from Invitrogen.

http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf#search=%22TOPO%20TA%22

Marina Fomin
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Tony Rook wrote:Marin Fomin:

Tony Rook wrote:

Marin Fomin:

Thank you for your suggestions!
I also thought to design a new primer set.

Depending on how important it is to keep you're designed primer set. You can either design a new set of primers or confirm you have only one amplicon through using TOPO TA kit (available through Invitrogen - these kits might be expensive) to perform a ligation to add several more nt to your amplicon. The appearance of two melting curves following this would confirm you're band data.

Concerning TOPO TA kit:
1) Do you think it is possible that I still have a single product despite several bands on gel?
2) As far as I understood this kit is for subclonning of PCR products. In the manual it is recomended first to be sure that you have a single product or to cut the write band from the gel. So how can it help in my case?

Thank you.

Tony Rook
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Marin Fomin:

Marin Fomin:

You are correct about the TOPO TA kit being for subcloning a PCR products. Someone had recently been using this kit in my lab and while brainstorming about your issue, I figured that this would confirm that one of your two bands was actually your desired amplicon.

You're best bet would probably be to redesign your're primer sets.

Good luck and please keep us updated.