Your primer sequences look good and the protocol should work.

Looks to me like you have way too much template DNA in the reaction. You shouldn't be able to see such a strong template band on your gel.

When amplifying material, I usually like to have less than 0.5 ng/uL starting template and you are currently at 3.6 ng/uL. I would reduce the amount of starting material 10-fold and then repeat the PCRs.

Dear Matthew Dunn,

Thank you for your reply! I will try with lower DNA amount. But one thing is I ran a negative control in the gel with all the samples together (didn't show above) which looks like the forth lane in the second figure. So maybe the big band is actually something amplified, not just the template itself.

Since my aim fragment is ~8kb, but even the big smear is above the 10kb ladder, which I'm really confusing because I blast my primers and it can not amplify that long fragment.

Have you done a long range PCR from gDNA? I'm glad to hear your experience about that! 

What do you mean by negative control? No polymerase, no primers, no template? If you don't see the template on the gel in the negative control, then that definitely doesn't mean it is a template problem.

Your primer melt temps are pretty high. I might even go with a two step PCR. 25x cycles of 95C for 30s and then 68C for 7-8 min. If that doesn't help, there are two additional strategies you can try: 1) cycle optimization, 2) touchdown PCR, and 3) nested PCR.

1) cycle optimization - it is possible that your PCR is working too well and you are over amplifying the material. That tends to result in a characteristic smearing above the expected band. To test for this, run the PCR at different numbers of cycles (i.e. 10, 15, 20, 25) and run them on the gel to compare. If it is over amplification, one of the earlier cycle points should look superior to the later ones.

2) touchdown PCR - by slowly reducing the melt temperature, you can force a more specific primer annealing to the expected region.

3) nested PCR - if it is nonspecific primer binding this strategy should overcome the problem.

Dear Matthew Dunn,

My negative control has only water and gDNA, which is the same amount as used for PCR.

I tried two step PCR at 25x cycles of 94C for 30s and then 68C for 7min+5s/cycle. You see the 4th lane, it's the result of two step PCR.

I will use the PCR product for sequencing, so it's convenient  for me to do it just in one step. But I will try your suggestion since one step haven't worked now.

Thanks!

An update of my PCR.

There are 3 buffers in the Roche Kit. They are for different length of PCR product. Buffer 1 for 0.5-9kb, buffer 2 for 9-12kb and buffer 3 for >12kb. Previously, I used buffer 1 because my aim product is 8kb. But today I tried buffer 3 and the rest condition didn't change at all. It seems that I got my band. I will redo this to confirm. Attached is my gel image.

Dear Shuaichen,

the Long Expand is a lazy enzyme. In my hands it takes 2min for every kB. So for a 8kB I would set 16min expansion and after 20cycles add another 0.5ul enzyme to the 50ul mix. However, I think for long fragments it is better to change strategy. First, use primers which PCR about 4kB of you fragment each i.e. 2 fragments; one 5kB and one 3kB will also work. These two fragments should overlap by at least 20Bp in the middle region of the genomic sequence you want to prime. When you get the two fragments, gel elute them. After gel elution, use equal amounts of both fragments as template in the same mix. The common middle region will anneal; hence it should be long enough to stick together at 68oC, the extension temperature for Long Expand. Use the 5' and 3' primers for PCR (no longer the middle primers), run a 1min annealing at 55oC and a 16min extension at 68oC. This will fuse both of your fragments together and since the fragments are the only template present, there will be no unspecific bands.

Dear Torsten,

Thank you so much for reply! I got one optimized protocol from one person and it worked for my PCR. She told me to make two separate reaction mix. Mix2 has enzyme, half of the total buffer and add H2O to 15ul. Mix1 has all the other things in 35ul.

I just put Mix1 in the cycler first and denature for 2min, then pause to add the Mix2. Then continue the whole reaction.

The attached is my gel image. The bright band is at the expected length. Hope the sequencing result is correct.

 Dear Shuaichen Liu . Please let me know complete protocol in detail. As i am also facing problem of smears with g DNA PCR. What taq u have used. Primer quantity, gDNA quantity pcr profile buffer total reaction volume etc. thanks

Dear Shuaichen Liu

I´m also interested in protocol. Could you share with us? Thanks

Hello, I also try Long PCR using LA PCR TAh¡kara Kit but I used a wrong thermalcycler protocol:

94°C 1min

94°C 30sec

20X 64°C 15min

72°C 15 min (10 or 6 kb frag expected)

72°C 30min

4°C ∞

Do you think, it works???

Thanks

Hi Shuaichen ,

Could you please share the protocol which helped you in amplifying the 8 Kb fragment

Hi Shuaichen Liu .

I am also trying the Long Range PCR.

Could you please share the detailed protocol that helped you to get the desired band.

We used ONE SHOT LA PCR

I use a conventional termalcicler.

The reaction was

1 denaturation 95º 1min30sec

2 denaturation 95º 30sec

3 annealing 64º (adjusted according to tm of the primer pairs) 7min 30 sec

4. Extension 68º 7min 30 sec (working to less than 7kb, increased 1 min ech kb).

Repeat 2 to 4 for 35 times.

5. Final extension 72 º 30 min.

you might need somes adjustement

Regards