Hi Adam,

PCR is fickle. Unless the specific PCR is totally optimized, not every PCR will work, even when they are technical replicates.

I ran the exact same mixture (mixed all in one tube) aliquoted across three 96-well PCR plates using 3 separate PCR machines (all the same brand, but 2 were older and 1 was brand new).

I did this to show my boss at the time (who would berate students for their PCRs failing) that a PCR working once doesn't necessarily mean that it always works.

What I could show were two things:

1) roughly 15% of the technical replicates failed regardless of machine; and,

2) the machines were not uniform in their thermal profile across all wells. In other words, they either didn't heat up or cool down uniformly, didn't all reach the same temp for equal amounts of time, or a bit of both.

There are two things I would suggest to decrease the technical replicate fail rate:

1) adjust Mg concentration to 2.5mM (go higher and you are likely to get non-specific bands popping up).

2) add DMSO to your mix (~ 0.5ul per 10ul reaction) - DMSO works far better than glycerol and it has been a standard addition to my PCRs for that last 30 years.

Depending on which Polymerase you use, the K concentration may also need to be optimized.

BTW, it has nothing to do with your agarose gel (concentration or voltage used), and 1ng template is no where near too much.

Good luck.

Dear Adam, the concentration of your DNA templates is so high. You can try it after dilute them and run slowly in gel, not too high voltage.

Looks to me like you have WAY too much template DNA in your reactions and you might also be over-loading your gel. How many cycles of amplification are you using?

Looks the DNA concentration of sample 3 & 4 is high. Try running at 80v for 45-50mins.

My question is; do you all run at the same PCR condition ?

If YES, troubleshoot the condition for sample 1, 2 & 5.

If NO, run all the samples at the same time and condition using Gradient PCR (to see the best annealing temperature). REMEMBER, annealing time and temperatture is very important in thermocycling activities !.

ALL THE BEST

All of you so far are commenting that it is too much input DNA, but I don't think that 1ng is that much is it? Could that explain differences in replicates? If I'm putting way too much DNA into the gel, then surely both replicates would give the same result?

Katie: I am doing 32 cycles currently, I had upped this from 30 because I was not getting bands with previous samples. For these samples, it looks like I'm getting a nice strong band (sometimes), so I could probably drop it back to 30. I'm loading 5uL of my 50uL of sample into the gel. I didn't think this would be too much, but I will try diluting?

Mohammed: The conditions are the same for all bands, it was all amplified together in the one block.

Dear Adam

First you have to check DNA concentration and decrease agarose in the gel for running , I prefer to do optimization in different concentration and number of cycle

Best wishes

Ahmed I Ahmed I have at various times tried 30, 32, 35 and 40 cycles.

I am doing 0.8-1.0% agarose gels, I don't think I wan't to go much lower.

I have since tried running my gel at very slow speed, and also diluting my PCR product before loading so as to not "overload" the gel. All I see is the same result. I can attach this if anyone wants to see.

I don't think this is an electrophoresis issue. I am sure the smearing is part of the PCR reaction. Perhaps there is some confusion; the replicates are PCR replicates, not just the one PCR reaction loaded into 2 wells gel wells. This is 2 PCR reactions from the same template.

To me it seems that the polymerase randomly struggles with this set of primers or my PCR block needs servicing. I'm thinking it's the former as a separate set of primers that amplifies a larger (11kbp) product seems to work just fine!

Actually, 1ng is not much for gel, as well 5uL of PCR product.

May be U further troubleshoot the PCR conditions.

ALL THE BEST

Hi Adam, weird I know, but could there be certain wells/rows in the block that are playing up? Can you try a different thermal cycler from a different lab? I've had something similar happen before, and it was the block

Hi Adam,

PCR is fickle. Unless the specific PCR is totally optimized, not every PCR will work, even when they are technical replicates.

I ran the exact same mixture (mixed all in one tube) aliquoted across three 96-well PCR plates using 3 separate PCR machines (all the same brand, but 2 were older and 1 was brand new).

I did this to show my boss at the time (who would berate students for their PCRs failing) that a PCR working once doesn't necessarily mean that it always works.

What I could show were two things:

1) roughly 15% of the technical replicates failed regardless of machine; and,

2) the machines were not uniform in their thermal profile across all wells. In other words, they either didn't heat up or cool down uniformly, didn't all reach the same temp for equal amounts of time, or a bit of both.

There are two things I would suggest to decrease the technical replicate fail rate:

1) adjust Mg concentration to 2.5mM (go higher and you are likely to get non-specific bands popping up).

2) add DMSO to your mix (~ 0.5ul per 10ul reaction) - DMSO works far better than glycerol and it has been a standard addition to my PCRs for that last 30 years.

Depending on which Polymerase you use, the K concentration may also need to be optimized.

BTW, it has nothing to do with your agarose gel (concentration or voltage used), and 1ng template is no where near too much.

Good luck.

most likely these discrepancies depends on the experimentator (just human) or the equipment (e.g. an imprecise pipette).

Hi, I agree with Megan Finch-Edmondson , the problem can be caused by the PCR machine. It happened to us when using somemachines with low consistency f temperatures (which means the annealing temperature is not same for all the reactions and then of course you get such results).