hi Yue,

it may not have failed but you may not have very much product as it is a long amplimer. try making 2 new internal primers...they do not have to be as stringently designed as the first set just inside the original 2 but can overlap the original primers by some bases as the target is now abundant. then run the nested PCR of 1/10th ul of the first round amplification with the new primers without purifying the first product. The number of cycles needed for nested PCR is quite low..I used to set up a few tubes and take them off the cycler at abou 5 cycle intervals from 10 t0 30 cycles

Another possibility is to consider  doing the PCR in 2 halves i.e PCR use 2 fragments of 2kb each. If you include an overlap in the middle of say 50 bp or more you can then mix the 2 templates together and reamplify a diluted aliquot  with the outer primer pair to generate the full length product,

Be careful of adding too much cDNA per reaction.  If your reaction size is 20 uL, this means you are adding 20 ng cDNA/uL.  The highest you should be adding (even if your cDNA target is rare within the milieu) is 2 ng/uL.  Adding too much runs the risk of shut the reaction down (template inhibition of the polymerase). Adding less is more in many of these situations.  Also, during cDNA synthesis, since your primers are possibly 4 kb apart, full-length cDNAs may not be synthesized by your RT enzyme - so one of the primers can't find home. Perhaps your 5' primer has no place to bind.

Paul, thank you! I tried nested PCR previously but didn't care that much on the cycle number of the second round PCR. I will try different cycle numbers to see what happens.

Hi Jack, thanks for reminding me that too much cDNA will shut down the reaction. I will reduce the amount of cDNA to 2ng/ul as you suggested.

Hi Lan, I thought about this method before. I thought 4kb was not that long, so I decided to try regular PCR first. If regular PCR still doesn't work, I will try this. Thank you!

Reverse transcriptase is a DNA polymerase with poor processivity.  If you use poly-T as the primer, most of the cDNAs will lack the 5'-side.  That's why RT-PCR is good for gene expression, but not very good to use as template for the whole gene, and 4-kb cDNA is a very long one.

Do you have no product or some not-specfic products?

Hi Amelia, in some cases I don't have any product, but I also have non-specific bands sometimes.

First of all I would design longer primers since 18 bases can recognize more templates (you can arrive till 60°C as annealing temp for each primer). Then I suggest you to check primers concentration: when you use a proof-reading enzyme (as Platinum) you need a higher primers amount since primers can be 'eaten' by the enzyme. Anyway before starting with a proofreading Taq I usually set up PCR with a standard protocol (just to be sure about reagents concentration and cycling conditions) and then I move to the high-fidelity PCR. Consider that it's common to have a lower yield with a proofreading Taq. I usually prepare a larger number of reactions. Finally, I agree about cDNA concentration: 400ng is too much, I would do 100ng (considering the big length of your gene).

Hi Amelia. Thank you so much for your suggestion. You said you will start with standard PCR protocol then move to high fidelity. Do you mean start with normal Taq polymerase then try Platinum Taq High fidelity DNA polymerase? or you mean the hot start PCR protocol? Thank you!

I meant with a normal Taq polymerase :-)

 Hi Lan, I am considring doing PCR in two halves now after many unsuccessful trials to amplify the whole fragment. I am wondering do I need to design the overlapping primers exactly in the middle of the gene, so I will get two fragments with same length, or it doesn't matter where I divide it? How many bp overlap will get the best result? Is 100 bp overlap too long? As for the third PCR, I will need to combine the two PCR fragments into one using as the template for the third round PCR? Is there any specific requirement on the condition of the third PCR, like annealing temperature and initial denature temperature?

Thank you!

Hello Ian Teo

I want to amplify 3500kb DNA using cDNA. When I tried using phusion PCR I am getting smear. So i want to try the method that you have suggested. Can you please send me the protocol link for that method.