You don't need a separate 2nd strand reaction. Your forward PCR primer will synthesize the 2nd strand during the 1st cycle of amplification.

You don't need a separate 2nd strand reaction. Your forward PCR primer will synthesize the 2nd strand during the 1st cycle of amplification.

when you quote the annealing temperatures of the primers is this the Ta of the part of the primer specific to the template or for the whole primer including the restriction site? Also have you checked that you have some cdna by amplifying a housekeeping gene fragment to ensure that your first reaction has worked. If your cdna ws produced with gene specific primers you might consider checking the whole sequence exists by getting a reverse primer at say 300 bases and another forward primer to match the original reverse primer at say 4500 bases. If both ends amplify then the whole molecule does exist and you can concentrate on the pcr reaction knowing your template is good

Dear Paul,

Tm I have mentioned is for the whole primer including restriction sites and few additional bases.

I will try amplifying housekeeping gene as well.

Hi Arun,

when you say that your gene size is 5kb, is this the length of the entire mRNA including a much smaller coding region (that you require), or is it the length of the (exon-free) coding region that you need in its entirety?

Secondly, do you know the sequence of the gene in question? The reason why this question is pertinent is that should the overall composition of the message be difficult to amplify end to end, another option could be to identify internal common restriction enzyme sites at (for instance) +1500bp and +3000bp, design complementary pairs of primers covering these restriction sites, and generate the full length sequence in the form of 3 1.5-2kb fragments that could be sub cloned into a PCR capture vector for sequencing and verified, and then assembled into the full length sequence by straightforward restriction-ligation.

Finally, again if the sequence is already known and the PCR continues to be problematic, a third option could be to generate the sequence by gene synthesis rather than rtPCR.

For the PCR, apart from using proofreading polymerase and the conditions cited, do you have any PCR additives present? Years ago we found that DMSO was helpful with PCRs that otherwise proved difficult, and there are a plethora of other components that can sometimes assist. Is the Magnesium content also normal?

Do you have any thoughts? Please feel free to discuss here further. Good luck, Robin

dear Arun,

you cannot use the whole size of a primer that has added non binding bases to calculate Ta. Only the part that binds to your target dna must be used for Ta calculation. If you use the whole primer sequence the annealing calculated temperature will be too high and in the first cycle the primers will not anneal so there will never be a product. Please recalculate TM for only those bases which can anneal to your target dna and try again at the lower annealing temperature ,

Only do housekeeping genes if the cdna was produced with random primers or oligo T primers Good luck

paul

I feel that the most likely cause of your problem is that the Tm of your two primers is so different. You say that one primer has Tm = 61 and the other 70. So using an annealing temperature of 60 is far too low for the primer with Tm = 70 to anneal properly. Check the following:

1. The Tm of your primers, excluding the restriction sites and clamp sequences should be as close to each other as possible. Use an annealing temperature that is 2-3 degrees below the Tm of your primers.

2. High fidelity polymerases polymerize more slowly than regular Taq. Typically 1 min/1 kb for Taq is OK but this may need to be increased for the high fidelity polymerase to 2 min/Kb or more

3. Check that your cDNA is good by using a bit of it to try amplifying a cDNA with primers that are known to be good, like if you have some primers your lab has used before, or try a housekeeping gene like beta-actin or GAPDH

4. Double check that the primers are in the correct orientation - forward primer has the same sequence as the mRNA sequence (AKA the published cDNA sequence) and reverse primer has the same sequence as the reverse compliment of the mRNA sequence - this is easy to get confused.

5. Also, as mentioned above, the Tm of the gene-specific part of the primer (excluding the restriction sites and clamp sequence) should be around 60 C, if the Tm of your primers with the restriction and clamp sequences is around 61 - 70 that means the Tm of the gene specific sequences are only around 45 - 50 which is far far too low!

Well Yoav has rightly pointed out that the forward primer will automatically synthesize the second strand. If you need to study it (I do not understand the rationale behind this) well you may do so.

Dear All,

Thank you for all the suggestions.

I have checked the Tm of my gene specific primer alone (restriction site sequences and extra bases are excluded) and the difference is more than 15 deg C. Now I have designed another set of forward and reverse primers whose gene-specific Tm are 59.33 and 59.12 respectively. But the primer lengths (without restriction site sequences and extra bases) are 19 and 30 for forward and reverse respectively. Is that fine if I design primers with such a difference in length? The additional bases will add 12 for both the primers. So 31 for forward and 42 for reverse primer. Any suggestions on this?

Dear Robin,

The 5 kb is the length of my entire mRNA of my gene of interest.

I know the full sequence of the gene. As you suggested, my next try is to amplify short fragments of the gene and clone the full length to my vector.

The GC content of the gene is around 45%. Do I still need to add DMSO additive?

Dear Göran,

As I mentioned in the above, the mRNA of the gene is around 5 kb. I set extension time of 1 min/kb for pfu enzyme. But I did not aware of the longer extension time is required for long gene. As Zachery mentioned, I will try to set 2 kb/min for extension.

Dear Paul,

Thank you for suggestion again. Now I have modified the primers as above.

Dear Zachery,

I have totally agree with you. I agree that the difference of Tm of my primers is high. I have modified the primers as above. I would like you to comment on the same.

Thank you so much for timely suggestions and I will update the results soon.

Arun.

Arun, the removal of template RNA can be critical for amplifying long cDNAs. This can be achieved with NaOH (~0.1N final) or enzimatially. You can find details of how to do this kind of digestions here: Article A long distance RT-PCR able to amplify the Pestivirus genome

Hope it helps.

What I usually do is design a normal gene specific primer first, that is only binding to your gene of interest, and then just add the required restriction site afterwards with 3 to 5 extra bases on the 5 prime end of your primer to allow restriction enzyme binding. Make sure your Tm for the gene spicific primers are around 60 degrees and try not to go past 29nt primer lenght before you add the restriction sites. Sometimes it is difficult, and you should not run into trouble here as I have used primers of up to 45bp long, but it increases the risk for non specific binding. Then when you do your PCR I usually use the Tm of my lowest primer as the annealing temp, this will work as you are working with cDNA so it is pretty clean. Never had any issues with this before.