Hi there,

Are you using exactly the same set of primers for both experiments on plasmid and mRNAs? If yes there might be a problem with the mRNA template you are using.

Hi,

the problem is maybe not your polymerase, if it is working for your PCR usually.

First of all Verify that your primers give you a band on DNA (is the size is not too big). This is important because sometimes the primers are not good.

Then verify the integrity of your RNA (do an electrophoresis, gel 1% agar, you should have specific band and not a smear) and verify the "purity" with a nanodrop.

Finally you should try to do a new cDNA library and cDNA with your specific reverse primer (maybe use more specific reverse primer). Sometimes cDNA library will give better result.

Sir what will be the problem with mRNA ?

Dear sir the Taq polymerase is working well with the other DNA templates. One more thing is I am using same forward and reverse primers for both the cDNA and the Plasmid positive control. The taq polymerase amplifies the target sequence from the plasmid but not from the cDNA. Further befor making the cDNA I have checked the integrity of the isolated RNA and also after the DNase treatment. The RNa is of good quality shows more than 3 distinct bands. Further I tried the cDNA protocol with the 5' adapter specific forward primer and 3' gene specific primer. There I havenot got any amplicon with the same target sequence specific primers. moreever the primers amplifies the entire gene which is around 750 bp long. Is there any damage for the 3' region will happen for mRNA. Could I use the reverse primer which binds inbetween the gene ? Because I am using the cDNA from 5' RLM RACE protocol.

Kindly give your suggestions !

Thank you,

I think you are correct try the second primer which is near to 3' end...or you could go for another 5'RACE.... to confirm what you are getting is your Gene of interest.

but i am not confused why you need entire sequence... for start site you are getting 5'RACE product. and if you want to know sequence you could have it  from plasmid.

 all the best ...

I have faced lots of problem when I am doing RLM-RACE from Invitrogen. Better I will get an idea when talk to you. Pls call me on this no 09840362493

I don't understand why you use an expensive 5' RACE kit to produce just cDNA, if the sequence of  5' part is known already, because you are using a specific fw primer (together with a reverse primer) . RACE kits are used for unknown transcripts from which only part of the sequence is known. You need to do nested PCR's with different (rev) primers together with the adaptor fw primer, to finally get your product of interest.

You should use a normal cDNA kit to make cDNA. After cDNA synthesis you can treat this cDNA with RNAseH to remove the mRNA, this helps amplifying difficult targets.

Dear sir, I thank for the kind reply. Sir I have to identify the 5' upstream sequence of my target transcript in N. benthamiana. For that reason I use 5' RLM RACE kit. In the RACE protocol, at the final stage the adapter ligated transcripts will be converted into cDNA. so the final cDNA will have the cDNA sequence comprising the 5' ligated adapter sequence followed by target transcript's upstream sequence. to amplify the 5' upstream sequence we should use adapter specific forward primer. I am using the same. In my case the taq polymerase doesnot amplifies the target transcript. but pfu polymerase gives some bands. kindly give some suggestions sir.

Now I understand better your problem, thanks for the update. Did you do a second PCR with a nested primer, or mayby even a second nested PCR with a different nested primer.

For the first step of making cDNA, did you use a gene specific reverse primer?

With this RACE kit only full lengt capped RNA is used, degraded RNA is not used, you could try the normal RACE kit that also uses degraded RNA as template. I tried both kits, but had a better recovery of the last one, but I should say that I used next generation seuencing to find full lenght sequences. With Sanger maybe the signal will drop down at the far 5' part. Also if your final product contains aspecific bands, next generation sequencing will give you the correct sequence when doing denovo assembly or looking by hand to find the right sequence out of other aspecific sequences.

Thank you sir. Your answers are very helpful for me. Based on the suggestions I have tried 5' adapter specific forward primer and 3' gene specific reverse primer for the cDNA synthesis. But This time I have not get any band. But i did some experiment with the previous cDNA that I prepared using Oligo dt. I got an amplicon very nearly to the expected size of my target transcript. So I have cut and eluted and again amplified using forward and Reverse primer and prepared sufficient quantity of DNA for further experiment. My target DNA will release 550 bp with Nco I and around 650 bp fragment with EcoRI. But there ie no release were found after digestion. Can you tell whether the PCR product will be digested easily by enzymes or it is difficult to digest PCR product after elution ? Further if the PCR product doesn't gives correct release of fragment means this may not be a DNA from the target transcript ! am I write ? Please give your kind suggesstions !

PCR products can be cut easily by restriction enzymes, you have to adjust the Mg2+ concentration to 10 mM. If high salt concentrtion is needed you can add some enzyme digestion buffer as well.

You first have to make cDNA with only your gene specific reverse primer, with a reverse transcriptase (RT) enzyme, after this you can do PCR with a DNA (Taq) polymerase, with forward (adapter) primer and a gene specicfic reverse primer. After this you have to repeat this (nested) PCR with anoteher gene specific reverse primer, this primer should be located 5' upsteeam of your first gene specific primer. After these three steps you can expect a product of your interest, if not you have to do a PCR again with again another gene specific reverse primer.