4 replies to this topic

[kshanik]

Dear all,

I am trying to amplify my gene of interest from cDNA. My primers are designed against the two ends, and I have been trying to get an amplicon of ~500bp. But till date I not got any product. I have tried a few different annealing temperatures [I am able to amplify other genes from the same cDNA].

Any suggestion will be of great help.

Many thanks.

[ivanbio]

Here are some suggestions:

1. Make sure your cDNA is of good quality and of high enough concentration. Alternatively make new cDNA in case the one you are using is degraded.

2. Design at least a second set of primers.

3. Obtain a PCR that is known to work and use it as a control to make sure all your reagents, and cDNA, are of good quality.

Cheers

[kshanik]

ivanbio, on 10 May 2011 - 06:30 AM, said:

Here are some suggestions:

1. Make sure your cDNA is of good quality and of high enough concentration. Alternatively make new cDNA in case the one you are using is degraded.

2. Design at least a second set of primers.

3. Obtain a PCR that is known to work and use it as a control to make sure all your reagents, and cDNA, are of good quality.

Cheers

Thanks Ivan,

I did make new cDNA, even got another cDNA from a neighbouring lab. Did not work.
From my cDNA I was able to amplify another gene, so it seems the reagents [except the primers] are fine. I also made a fresh working stock of primers to address this issue. Still not-a-thing!!!

I dont have a lot of room to design primers since I want to amplify the whole gene. The primers that I currently have are complementary to the gene ends and also have restriction sites in them. I am thinking of getting a new set of primers without the RE site overhang: amplify the product first, and then use the product as a template to incorporate the RE sites!!!

But any other suggestions before this PCR drives me crazzzy...

Thanks a lot, Sanjita

[dpo]

I have some questions that can help you to troubleshoot:

Do you know if your gene is expressed in the cell line/tissue where you got the cDNA from?

Can you amplify the fragment from genomic DNA?

Which polymerase did you use? And do you have access to others?

How GC rich is the fragment?

If you can answer these, maybe we can suggest some other options to try.

[kshanik]

dpo, on 18 May 2011 - 01:16 AM, said:

I have some questions that can help you to troubleshoot:
Do you know if your gene is expressed in the cell line/tissue where you got the cDNA from?
Can you amplify the fragment from genomic DNA?
Which polymerase did you use? And do you have access to others?
How GC rich is the fragment?

If you can answer these, maybe we can suggest some other options to try.

Let me reply to your questions:
>Do you know if your gene is expressed in the cell line/tissue where you got the cDNA from?

Yes, I know that my gene is expressed. I have used the same/similar cDNA to check the levels of this gene in qRT.

>Can you amplify the fragment from genomic DNA?

Never tried that, the total gene size is ~1.5kb with multiple introns. I just want the cDNA [which is about 550bp].

>Which polymerase did you use? And do you have access to others?

I tried various polymerases. Taq, Pfu and Phusion. If you have some other suggestion, I will see if I can "beg/borrow/steal" it from somewhere.

>How GC rich is the fragment?

It is about 45% GC.

Let me know what I can do. Thanks a lot.