4 replies to this topic

[novagen]

Hi All,
how do we differentiate forward and reverse primer in a pcr reaction.
I have used two different reverse primers to amplify the end region of a gene. which strand of the DNA would have been amplified and how ?
Iam unable to get a clear picture of it
any explanation is appreciated.
Thanx

[little mouse]

you need one forward and one reverse to do an exponential amplification
you only did linear amplification. not sure you can see something

[cellcounter]

novagen, on Jul 3 2009, 06:30 AM, said:

Hi All,
how do we differentiate forward and reverse primer in a pcr reaction.
I have used two different reverse primers to amplify the end region of a gene. which strand of the DNA would have been amplified and how ?
Iam unable to get a clear picture of it
any explanation is appreciated.
Thanx

1. As above, if you have used only reverse primers in the reaction.

2. If you have used one Fw and two Rev primers simultneously, you should see two products in one reaction.

3. If you have used the Fw primer with one Rev primer, each, in two individual reactions, you should see one unique product in each reaction.

As to your specific question:

Fw primer binds to the antisense strand, and amplifies a new sense strand from it. Rev primers bind to the sense strand, and amplifies the new antisense strand.

[novagen]

cellcounter, on Jul 4 2009, 05:05 PM, said:

novagen, on Jul 3 2009, 06:30 AM, said:

Hi All,
how do we differentiate forward and reverse primer in a pcr reaction.
I have used two different reverse primers to amplify the end region of a gene. which strand of the DNA would have been amplified and how ?
Iam unable to get a clear picture of it
any explanation is appreciated.
Thanx

1. As above, if you have used only reverse primers in the reaction.

2. If you have used one Fw and two Rev primers simultneously, you should see two products in one reaction.

3. If you have used the Fw primer with one Rev primer, each, in two individual reactions, you should see one unique product in each reaction.

As to your specific question:

Fw primer binds to the antisense strand, and amplifies a new sense strand from it. Rev primers bind to the sense strand, and amplifies the new antisense strand.

I have used two different  reverse primers for amplification.How do i support this

[uncutlateralus]

Forward is 5-3 and reverse is 3-5 and thats it.

not sure what you mean, however.

if you get some primers and not sure which is which just get the target gene your after and just BLAST the target sequence with your primers to see which is which, remember to do this complementary aswell just to be sure.

If you've used to DIFFERENT reverse primers to do a PCR sequentiually then it may be that your doing a 'nested' pcr.

In terms of why you do this and explaination a simple text book will tell you everything you need to know, the 'ins and outs' of a pcr reaction is very basic level molecular biology.