It may help for you to explain and provide evidence for "Does not work". What results show up on a gel after running this PCR? Are your primers absorbed at all? Are you generating unspecific products?

A classic problem in PCR is poor template, which is especially true if many other reaction conditions are changed and no product results. How do you know your cDNA contains the full 10kb fragment you want to amplify? You mention that the cDNA you used works, but how have you check this? I am no expert on RT, but I believe getting cDNA of 10kb is quite tricky...

Like Geno said, 10kb is not easy. Are you starting from total reverse-transcribed cDNA, or a clone of the specific cDNA? The former will be much more difficult than the latter. It sounds like you have tried a lot of trial-and-error optimization, which is good. You could also try slightly different primers. Finally, it may help to make a few primers that are designed to amplify smaller portions, like amplify two overlapping 6kb pieces, or even smaller. This will tell you if your template is in good condition and that the target is actually there.

....and don't forget to increase the extension time of your reaction. An old "rule of thumb" is to extend 1 min for every kb. This would give a very long extension time though for a 10 kb insert!

Seems the primer Tm is very low?. If you can re-design it with Tm near 65 degC, you can also get PCR faster. This could also help for better RTase reaction.

thank you all for the feedback. I tried using genomic DNA. but i could not find any bands. I did cDNA synthesis from mRNA and used it as template. I did get a band around 10kb first but same conditions and Tm i tried next time, the bands dont appear. Only the non-specific band around 5 kb. I have attached herewith a ppt file that shows my cDNA works and 10kb pcr conditions and gel picture. 

Try Klentaq LA from DNA polymerase Technology

How do you generate your cDNA? Many of the RT enzymes available would have problem running a full 10Kb so you may need to generate your cDNA in fragments, maybe use a mix of gene specific primers spaced every 1-2Kb and then try to amplify it in several segments like Michael suggested although I would suggest 3x4Kb  or maybe even 5x3Kb and then do the assembly in 2 parts first 2x(3x3Kb) and then the full 10Kb.

Dear Mr.Lubelsky,

Thank you for your valuable suggestion. i used the Takara cDNA synthesis kit with oligoDT primers. Like as you had mentioned, if my cDNA does not have entire 10kb segment in it, when i try to amplify as shorter fragments of 3kb 3kb and 4kb i get PCR product  as mentioned in the ppt file .This is nothing but 10kb fragment divided into three parts. kindly help me with this. 

Extensor mastermix worked well for me for 12kb frags. Its from Thermo Scientific. I comes as readymix for

Your tests for fragments of your gene by priming short segments are not total proof of full 10kb cDNA, but they are not a bad indicator. If we assume you have good cDNA a few issues still stand out to me.

First, the polymerase you use doesn't seem optimized for such large fragments. The manual provided with KAPA states:

The KAPA Taq PCR system is suitable for the amplification of fragments up to 3.5 kb from genomic DNA or 5 kb from less complex targets. 

Although this may be a concern, I wouldn't believe it to result in no product at the appropriate size. I would also not recommend using a polymerase without proofreading for cloning purposes. I usually clone with 1:1 taq:pfu. But, again, this isn't the cause of not generating a product. 

Next, you list this as you reaction mixture:

D/w - 14.4

10 buffer KAPA BUFFER - 5 ul

dNTP mix (10mM) - 1 ul

HT29 cDNA – 1 ul

Fw – 1.5 ul

Rv – 1.5 ul

Kapa – 0.6 ul

Total 25ul

You seem to be using a 2x concentrated KAPA reaction buffer in the final reaction. If your PCR total volume is 25 you need 2.5 ul of 10x KAPA buffer. 

My next concern (as mentioned by others) is the rather short elongation time you are using. You state 5 minutes elongation, the KAPA manual states 1min per KB, which is a normal estimate for Taq based polymerases, try increasing elongation to something closer to 10 minutes.

If I understand your diagram in the .pptx file you are using an annealing time of 0.15 minutes, meaning 9 second? This is very short even if your primers are quite small; I have never used less than 20 seconds for annealing. Lastly, the denaturation time in your loop is stated 0.20 so 12 seconds (60*0.2)? This is too short of a denaturation time for a 10kb fragment. 

Since you didn't state the concentration of your primers or cDNA I cannot comment on this. 

Best of luck, 

Geno 

Dear Villafino,

Thank you for the detailed explanation. I would like to mention that the primers and the cDNA were taken according to the concentrations mentioned in the kapa protocol. and the pcr machine i used is  biorad company. So the time mentioned for extension and annealing is all in seconds. I had mentioned by mistake that the buffer is 10x, its actually a 5x buffer  which i add 5ul to a 25ul reaction as given in protocol. This kapa polymerase is for amplification of longer fragments. The extension time mentioned is 30 sec per kb in protocol. But i even tried 1min per kb as you had mentioned. I am not able to trace out what other conditions can be changed. It will be very grateful if you could kindly suggest me. Please find attached the protocol for the kapa polymerase i use. 

Dear Quinlivan, 

Thank you for the suggestions. i will check this. 

I would try using Q5 polymerase or Phusion... Reverse transcriptase will not be able to make a 10 kbp amplification, usually it falls off much earlier only giving you partial fragments. 

A genomic library with restriction sites may be an alternative? Or doing 2 PCRs and joining them (Gibson assembly/utilizing internal native restriction sites, etc?)

You can amplify it using the TAQ polymerase from TAKARA

TAQ polymerase from TAKARA

Use the Extensor mastermix from Thermo, I've amplified many frags between 6-12kb