Try first lower annealing temperatures (usually 55ºC). This first step allow you to check if it is possible to see some bands. In case you find several bands, you can rise your temperature up until unspecific bands dissapear (not always possible).

When cloning into pDONR221 (with long primers with ATTB sites) the PCR is not always easy and 62-74 degrees is very high (note that the annealing temperature must be according to the part of the sequence that anneals with the gene, not the entire primer).

My advise, try 50-55ºC. You'll get bands if evertything's OK (use a positive control if you can), If not, design a short primers (without ATTB sites), amplify and then repeat PCR with long primers (like a nested PCR)

All the best

Try first lower annealing temperatures (usually 55ºC). This first step allow you to check if it is possible to see some bands. In case you find several bands, you can rise your temperature up until unspecific bands dissapear (not always possible).

When cloning into pDONR221 (with long primers with ATTB sites) the PCR is not always easy and 62-74 degrees is very high (note that the annealing temperature must be according to the part of the sequence that anneals with the gene, not the entire primer).

My advise, try 50-55ºC. You'll get bands if evertything's OK (use a positive control if you can), If not, design a short primers (without ATTB sites), amplify and then repeat PCR with long primers (like a nested PCR)

All the best

Thanks Alberto. I am going to try it.

Hi Anshika,

I would agree with Alberto. It sounds like the annealing temperature for the PCR is set to high. At 73 degrees, you would be annealing above the extension temperature. I would also start with a 55 degree annealing temperature to see if you get amplification. You can always raise the temperature later during PCR optimization.

You could also try touchdown PCR and start with a higher annealing temperature (maybe 68 degrees) then reduce 1 degree for each cycle of PCR until you reach the target annealing temperature of 55 degrees. This could reduce the number of false products. However, I would not anneal at a temperature higher than the extension temperature.

Good luck.

Lowering the melting temp didn't work. I still see no bands

1. How about the primers' GC content? Too much GC also hinders PCR amplification; Design the primers with less GC pairs.

2. How's the DNA template, Genomic DNA or cDNA or plasmid contained DNA? For the genomic DNA, try to dilute it by 100X and it may work.

I am using the cDNA as a template and GC content is less. I am going to use both high fidelity DNA polymerase and Taq polymerase. Hope it show up some bands.

I've done a lot of trouble-shooting for difficult PCR's recently. Here are the things I had to optimize: (1) Tm - you should do a gradient with your expected optimum temperature in the middle. (2) Try increasing the concentration of MgCl2, this sometimes helps for difficult to amplify sequences. (3) You can add DMSO to the reaction. This helps for high GC content regions or some otherwise difficult regions. Both this and (2) will require you to do a temp curve to find the optimal temp. You can pretty easily run one big optimization PCR with parallel +/- extra MgCl2, +/- DMSO, and a gradient all at once. (4) Increase or decrease template. Shouldn't be a problem for cDNA, usually only a real issue for genomic DNA (high genomic can inhibit the reaction by binding your primers at non-specific sites). (5) You can always try a different polymerase. Sometimes for whatever reason one brand works better than another. I recently spent a good two weeks trying to optimize with KOD polymerase and it worked the first time (with a temperature gradient) with Phusion. Finally, (6) primers. Sometimes you just need new primers, no way around it.

Hi Simon thanks. I would appreciate if you could send me the protocol (reaction mixture and per conditions) for amplifying the larger gene fragments

The protocol will depend on what conditions work for you. It's not really possible for me to predict what will work for you but if you tell me the Tm of each primer and your GC content (both for the primers and the region amplified) I can make a suggestion. Mostly optimization is just empirically derived but you can make some educated guesses. My first guess is that your annealing gradient should be lower (unless you've got really big primers) and your annealing time should be lower (for Phusion, for example, it is only 10-15 sec). Extension time for Phusion is lower too - 15-30s/kb works well. Do you know that your template DNA is good and that your polymerase is good? Have you thought about using a different polymerase? Are you sure the primers were designed off of cDNA and not genomic (ie not spanning a splice site?). How many cycles are you running? What is your actual polymerase and what are the suggested cycling parameters? A product number would be helpful so I know exactly what you are working with.

Simon's suggestion is very helpful for the beginner. MgCl2 and DMSO will definitely improve PCR amplification, however for long chain PCR, how's the PCR amplification fidelity? Have you sequenced your product?

I have designed the primers (both forward and reverse) of 21 nucleotides and they have the ™ of 62C. Before both the primers I have also added attB1 site to forward and attB2 site to reverse further followed by start codon in forward and stop codon in reverse. So my Forward Primer is now attB1+Kozak sequence + 21 nucleotides primers to gene of interest. Similarly Reverse Primer is attB2+stop codon + last codon as AAA + 20 nucleotides primers to gene of interest. Now these primers have the ™ of 83C. I am using Phusion High fidelity DNA polymerase from NEB.

I designed the primers using Primer 3 and taking mRNA sequence as the input query for giving out the appropriate primers for my gene of interest. Further I checked the primers using ucsc insilico pcr.

I am using the following pcr conditions 98 for 30 sec; 98 for 10 sec, 52-58 (temp gradient) for 30 sec 72 for 6 min and 72 for 7 min and ending up at 4.

I think I should consider the ™ as in between 58 to 64C as these are the for gene of interest and not the 83C. Please suggest

If you have primers pairs of 25-45 in length with melting temperature greater than 78 C, try the annealing temperatures of 52 C, 53 C and even 57 C seems to be ok. I successfuly got the amplification of my 9kbp plasmid DNA using primers with 36 ntd in length with a melting temperature of 78 C. i have used the annealing temperature in the gradient 52-57 and it worked for 52 and 53 C.

My primer length is 58 nucleotides for forward and 56 nucleotides for reverse

OK - how much of it actually anneals on the first round of the PCR? You are adding overhangs on but you should plan your PCR to account for the annealing temp of the segment that actually recognizes your sequence of interest from the cDNA. Sometimes you can get away with using that lower Tm for the entire PCR, sometimes it works better to do a few cycles at the lower Tm and then increase to the higher Tm for the remaining cycles. DMSO will also help with annealing, but it won't rescue the PCR if you've set your annealing temp to the entire primer.

I didn't get this how much of it actually anneals on the first round of the PCR? So my primers ™ is 62 C so I am gonna put the temp gradient 58 to 66. I am also going to use DMSO 3% of the reaction mixture, Taq polymerase (0.8) + Phusion high fidelity Taq (0.2). Using template as cDNA 3ul (300ng) 0.2 ul of MgCl2 10mM of dNTPs, 10pm of each reverse and forward primer 5X fusion buffer and water. Hope it will work this time.

I don't know if it could be inappropriate what I'm going to say. The reverse primer must be in reverse complement. Did you notice this thing? I suppose so, but I want to help you and maybe, designing the primers you forgot to do it.

If the last 20 nucleotides + STOP of your gene are:

aaatttgggcccaaatttgggTGA

Your design must be:

GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN-TCA-cccaaatttgggcccaaattt

In N you can put whatever, avoiding a STOP codon.

My best results were given by using iProof (BioRad) polymerase. But, any High Fidelity Polymerase must be OK. I don't think that's the problem.

All the best!!

Hi Anshika - you are adding extra sequence onto your amplicon for later use with the cloning kit, right? Your primers are partly made up of sequence matching the cDNA and partly of the add-on sequence, correct? I'm just asking if your reported Tm is for the entire length of the primers or just the part that is complimentary to the cDNA. Given the length of your primers it seems like you're accounting for only the complimentary portion.

Albert - I don't know why a stop codon would have any effect on a PCR reaction - DNA polymerases don't stop at stop codons.

Hello Simon, The ™ for the primers that is complimentary to cDNA is 62 (calculated from Primer3). The ™ for entire length is 83C. I am using Phusion High fidelity DNA polymerase from NEB so they uses higher ™ so I calculated the ™ on their site and put the temp gradient from 65 to 70 C. I haven't check the bands as the reaction is still going on but lets see.

I know DNA polymerase do not stop at stop codon. I'm only describing gateway cloning protocol and at N position I usually include some nucleotide not generating STOP codon. In this case, it's not a problem, but it is in forward primer design (NN position cannot be AA, AG or GA). Sorry if I didn't explain it properly.

after putting the PCR with the above conditions I just see the bands of my unused primers and nothing else so now I am going to do cDNA synthesis. May be some problem with the cDNA as I don't think there is some problem with the primers. PLease suggest

If cDNA is a problem, you can run RT for 1~2 hours since longer incubation time can increase the efficiency of reverse transcription.

I would suggest you to go for LA Taq from Takara. When everything failed, I have seen it gets past in amplifying longer and difficult regions.

I would suggest using a polymerase that is specific for amplification of a large PCR product. I was trying to amplify a gene of similar size and only got the desired product of around 7 kb when using KOD Xtreme polymerase (Novagen). Before switching to this polymerase I spent a long time trying to optimise my reaction when using Phusion high fidelity polymerase and when using Qiagens Long range PCR kit. The KOD Xtreme polymerase is very good and worked first time for me. I hope this helps.

Hi, if you are using PHUSION (Finnzyme) then denaturation should be at 98 not 94, extension 15/30 sec/kb, I would suggest check your cDNA using internal primers and also reduce your cDNA concentration. Primer shouldn't be a problem if you designed using Vnti or Geneious. Some of my GATEWAY primers are 60 bp long with Tm of 85 but works fine.

For 6,9 Kbp increase extension time to 9 minutes, this is the only way it will work.

I recently amplified 6.5 kbp. I also found that brand of the polymerase is important, and optimal one can be different for each specific case. In my case, high fidelity EasyA from Stratagene worked, while other polymerases didn't. The other thing that I found is that I needed to decrease the Tm, as compared to the theoretical one, so if the theoretical tm was 60-65°C, with 55°C it worked much better. I had some aspecific, but I could purify the right length insert from agarose gel. Finally, I also used little higher extension time than you, around 7 min for a 6.5 kbp. This is polymerase specific, but many polymerases require 1 min/kbp. Good luck!