Dear Hoan,

amplifying 9 kb by PCR is not an easy experiment, the dNTPs might run out before the PCR process completion and the template could re anneal to itself before the extension happens so your primers need to be anneal at very high temperature to minimize the time that the template has to become double stranded again. you might need to design primers with annealing temp 72c and try doing the PCR with1Molar betaine to keep the template melted for longer by destroying the triple hydrogen bonding of C-G so makes the template melt easier. if your current primers annealing at 53c or near, then it is very low for long PCR!

Good luck!

Dear Hoan,

amplifying 9 kb by PCR is not an easy experiment, the dNTPs might run out before the PCR process completion and the template could re anneal to itself before the extension happens so your primers need to be anneal at very high temperature to minimize the time that the template has to become double stranded again. you might need to design primers with annealing temp 72c and try doing the PCR with1Molar betaine to keep the template melted for longer by destroying the triple hydrogen bonding of C-G so makes the template melt easier. if your current primers annealing at 53c or near, then it is very low for long PCR!

Good luck!

how many cycles are you running the second time?

10 cycles is 1000 times more product and 20 cycles a million times more so if you run too many cycles then the product anneals with itself and just forms long smears not bands.

I would dilute 1/1000 and set up 6 pcr tubes and remove a tube at 12,14, 16 etc cycles and see what gives you a good band. I would normally add betaine to 1Molar and dmso 5% to help but if your first pcr works then obviously use the same pcr parameters for the second pcr

If you put the first template as a positive control and now it did not work then something changed in your experimental setup. What changed and how is not always easy to find; the most effective solution would be to find someone experienced in your institution and ask directly for advice.

Good luck!

do you need the entire 9kb? Nested primers just inside ( well the 3' end anyway) of the outer primers often work very well for re amplifications usually re amplifying only the desired amplimer

I agree with Alejandro Martin. If you used the same template (where the first PCR used and successed) as a control and had no results, this is very obvious that something have changed for the second PCR and lead to no product produced. You should worry about why even the successful template did not work. Do you need to re-amplify your first PCR product to do the next experiment?

Eman Bassiony : Thank you for your answer. The key point here is that I have included the DNA template for the first PCR as a control and it does not working now. The annealing temperature of my PCR is 70 degree.

Paul Rutland : Thanks for your suggestion. I run 30 cycles for the 2nd PCR. I am not sure if the number of cycles is the problem because in that condition, the control PCR should work as it worked well before. In addition, I need the whole 9kb as that carrying the gene I would like to clone.

Alejandro Martin : Thank you for your suggestion. This problem happened to me twice before and we could not find the solution for that, it is so mysterious. I even ordered primer again and again, try to change the reagents one by one but they were still not working.

Yuan-Yeu Yau : Thanks for your answer, I agree with you about the things we should consider.

Couple of things you might check (remote troubleshooting is hard, so my apologies if these are obvious):

One often overlooked source of variation is uneven heat distribution across the blok of the thermal cycler. Have you tried using a rotor-based cycler instead?

Also, as someone (I think it was Dr. Basiony) mentioned above, dNTPs are important. Some brands are contaminated with significant amounts of dUTP, and some polymerases -especially those used for long PCR- are fickle about dU-containing templates.

Hope it helps!

If I am reading your question correctly, you have a PCR reaction that worked. Now when you try it again it no longer works, and it does not work using product from an earlier that had worked? If that is correct, then the issue would seem to be a component in your PCR reaction, the conditions have changed or the machine has changed. I would try a control reaction first with entirely different primers and templates to rule out a problem with your PCR components. Make sure nobody accidentally changed your program on the PCR machine, and just work through using appropriate controls all the different variables. There is no obvious simple answer.

Hi, all.

Dear Hoan Dinh, I do agree with all support you got here and would only add the possibility of amplicon contamination (If you do not test it or consider it yet).

I'd ask some else in the lab, using the same mastermix but from another batch not used by you, to test your first reaction and then perform a second one as you did, and see what happens. Ok?

Amplicon contamination is a hell of a problem.

Hope it helps.

All the best.

Dear all,

Thank you for your valuable suggestions. I am still struggling with that issue.

I have tried another pair of primer with the reagents I used for the PCR that worked before and it works (same DNA template, just use different primers). Therefore, I do not think the contamination in those reagents is the problem here.

I have consulted the senior researchers in my institution, and learned that the primers are sometimes not durable by itself, in the other word, there was something wrong happened within the working solution (and the stock) of primers when stored in the fridge and freezer (4 and -20 degree celcius respectively).

I do not want to redesign primers but it seems that I have no choices as my studies cannot last for ever.

Best wishes,

Dear Hoan Dinh.

It maybe one possibility but, honestly, as your first reaction was right, It doesn't seem to be the case (if you're doing the second reaction with the same primer pair). If the problem is there, probably, It would be connected to the primers dimerization as in your second reaction you will have much more primers to dimerize.

Instead, I'd do what @ Paul Rutland told you to do. At least If you did perform a long cycle (35 - 40 cycles). In the case you did, decrease it to 25 - 30. Or as he said, do your PCR with 40 cycles but take tubes in different cycles and do a gel with all of them to check in what cycle you still can get nice results ...

Another possibility would be to make a gradient and check if any other temp (more restrictive) works better. May be the case.

If you wish us to give better opinions, we would need more info ... If it is not a problem for your work to give us the info.

Primers sequences;

cycling conditions;

mastermix used (with all details enzyme concentration, Mg salt concentration, dNTPs, etc)

Every detail counts.

All the best for you and your project!!!!