Hi Prabuddha,

After u have made the cDNA, chk with particular internal primers, to chk whether the transcript of ur gene is present in the pool of cDNAs u synthesized. If it is present then go for more primer combinations. If u dont get positive result with internal primers then there is problem with the tissue u have chosen then try for some other tissue in which the gene of ur interest is abundant,

All the best

Regards

Mahalakshmi

Hi Prabudda,

I am very much interested in your research, although it is far beyond anything I have ever done. Are the oligonucleotide primers, those that are best suited for your tissue sample of study? Sometimes primers can bind with other than the sample of choice. Additionally, when I have worked in the aDNA, I have had better results using Deep Vent rather than Taq polymerase.

Best regards,

Janice Cori Cobb

Thank you Mahalakxmi. Actually I have already obtained the sequence of the 3' end of the gene by 3' RACE from this fungus. No data is known for the gene from this fungus. So after obtaining the 3' end it is already known that the gene is present. Then when I am trying to do 5' RACE, I am facing the problems.

Tnak you Cobb for your response. Actually I have already obtained the 3' end of the gene as I have said earlier. Then I have designed primers from the fragment. So it is obvious that the primers are specific. So it is not the issue. I have used stringent melting temp. for the PCR also. but....... Anyways. Thank you again. And hope to get more suggestions from you people. And deep vent is a hi fidelity polymerase, isnt it?If my primers are specific. Then what difference the proof reading enzyme will make? do you think it will? Please elaborate Cobb. I am in a hell lots of problem here. Thanking you.

Go for some other primer pairs, design homology based primers, if you have this gene in any other species as well. Also, 5' RACE is bit more difficult than 3' RACE. If u r not particular about cDNA, u can go for genomic DNA and try techniques like genome walk etc. Hope this wud b of some help.

Regards

Mahalakshmi

H Pradudda,

Deep Vent "R" (as opposed to the original Deep Vent), has 1.5 times the fidelity of taq, a 1/2 life of 23 hours at 95C, and of 8 hours at 100C It works great for complex loop procedures and also for primer extensions. The native organism from which it is derived (pyrococcus GB-D) was isolated from a submarine thermal vent (of 2010 meters), and is able to grow at 104C!

I had used Deep Vent original to successfully recover aDNA from dentin of 3,000 year old Chinese dental remains (lots of exogenous contaminants and degraded sequences).

Best of luck to you!

Janice Cori Cobb

Thank you Mahalakxmi.

Actually, I have checked all my primers, they are working fine. I have designed them from the 3' RACE product and did PCR using the 3' RACE cloned product as the template. They are working fine. but what is happening in case of 5' RACE I dont know. Anyways. Genome walk may help. Thank you for that. Can you give any detailed 5' RACE protocol? as well as genome walk protocol which is a bit easy and you or somebody you have seen, have used before and worked?

ragards

Prabuddha Sarkar

Thank you Cobb. i dont have Deep vent in my lab. but I have pfu DNA polymerase with me. Will this help? Are these enzymes specific and accurate than Taq? then I may use them. Thank you again

regards

Prabuddha Sarkar.

I have done genome walking and have got positive results as well....So I can help u out with it.....Actually there are many protocols available.....I have used adapter based method. I can mail u the paper based on which we started with genome walking....or else now we go for Clonetech Genome Walker Kit, which is costly though but working in our system. Or else u can read through its manual n get an idea how to go about and then design adapters according to the paper which I would be able 2 mail in a day or so. Pls gv me ur mail id n I will send u 2morrow.

Hope it works in ur system too.

All the best

Regard

Mahalakshmi

Thank you Mahalakxmi,

My E-mail id is [email protected]

Regards

Prabuddha Sarkar

Sorry prabuddha I unfortunately dont have the full text of this paper. But u can note down the paper reference as follows, if it is online available there:

An improved PCR method for walking in uncloned genomic DNA. Siebert et al. Nucl. Acids Res. 1995; 23: 1087-1088. If u get one do let me know. Did u try to find out the Clonetech Genome Walker Kitand its manual?

All the best

Regards

Mahalakshmi

Actually I also dont have the access. Anyways, Thanks. I'll try to have the kit manual. The concept I can understand. Thanks a lot. What subject are you in?

Regards

Prabuddha Sarkar

I m into Plant Developmental biology....wht about u? I would try 2 scan u the paper n then send if u cant understand by the kit manual.....So r u planning to "walk" or "race"...lol....Do let me knw if u face any prblm.....

All the best

Regards

Mahalakshmi

Hi Mahalaksmi,

Thats very kind of you. I am trying to clone a gene from a fungus. I am planning to at first RACE, then when I'll become tired, I'll walk. lol...Actually I have posted a comment in your thread stating RACE where you have said about primer design. please check it. Another thing is that, if I am not getting 5' RACE, then how is it done? I may buy Ambion RACE kit. It may help I think. Anyways. Thanks again

Regards

Prabuddha Sarkar

hi every body ,

I want to isolate a promoter of gene, specific to perticular tissue. but i am facing problem in amplification of upstream of the known region. your suggestion are most wellcomed for helping me

Hi, Pankaj da. I ma aslo facing the same problem. I am trying to isolate a gene from a fungus. But the primers are not working I think. by the way, Do you know Vinod Kumar Gupta? He is my senior here. Anyways. Thanks

Regards

Prabuddha Sarkar

This is the article you are talking about 

Hello Sarkar and everyone else.

I'm experiencing the same problems with my 5'RACE PCRs.

Did you find out the problem?

Any solution or recomendation?

Thank you in advance.

Hi Daniel, Sorry for thee late reply. That project was completed and published already.