Hi and good day to you. I have also faced the same problem and believe that it's because the material of some PCR tubes is of inadequate quality and should have a minimal heat resistance compatible for use with standard thermal cyclers. I tried several methods which include: 1) using another brand of PCR tube, 2) sealing the tubes with parafilm or aluminum foil; 3) using another PCR thermal cycler; . For method 2, although the tubes will still melt, they were covered tightly with the parafilm or foil to at least prevent the tube contents from drying out. May you find these methods helpful or trigger a better solution to this problem.

Is a volume of these tubes 0.2 or 0.5 mL? In the latter case, the tubes could not be suitable for the thermal cycler.

1. Calibrate your PCR machine. Probably, due to continuous usage, the temperature program has got affected which increasing the temperature of the well than the allotted one.

2. Replace your PCR tube also, it is a tube quality issue.

Tan Suet May Amelia Igor Oscorbin

I have changed the tube brand today and running protocol in a different thermal cycler, let's see what's gonna happen. The vol i am using is 0.2ml

I need another answer, If there is is smear/tail formation in gel results, is it possible that the DNA i am using has deteriorated or it got contaminated, i got different results in 2 days. One day there were band and another day there was tail /smear formation. Or There could be possibility that there can be a issue with primers. I hope i may get some proper solution.

If you have changed the tube and still facing smear issues then recheck your RNA quality and if possible synthesize cDNA again. Further, check the housekeeping gene first. I believe that you are knowing the standardized Tm of your primers.

PCR machine calibration, or change your tubes

We have some Eppendorf epgradient PCR machines and their automated lids squash the tubes if you only have a few samples. We find that putting your reaction tubes in the middle of the block and a few empty tubes surrounding them (e.g. B1, B9, G4, G9) takes some of the pressure off the centre tubes and stops them compressing. Having said that, the reactions usually work even when they're a bit squashed.

One other thing to check - some blocks have holes for the 0.2ml tubes as well as smaller holes surrounding them (example image attached - BioRad says it's to reduce the mass so the block heats and cools faster). You're not putting the tubes into the smaller holes are you? Best of luck.

I would suggest:

1. Use good quality PCR tubes.

2. Keep lid temeprature just 2C higher than your highest temeprature.

3. Set the time of 7th step (4C) to Maintain at 4C to atleast several hours, so that while the PCR tubes are in PCR after the program completion, temperature won't rise to lid temeprature again.

Regarding smear, it can be due to high DNA concentration, so try dilututing your template prior to its use in PCR.

Good luck

I agree that these tubes look crushed, rather than simply melted. If they are 0.2 mL PCR tubes, and you are placing them in the correct holes, then i also think your problem is overtightening of the lid. The C1000 touch has a lid which is manually tightened using a turn knob which you are meant to turn until the ratchet stops you tightening any further- the pressure at this point might be too much for your tubes, so try loosening the lid a little before cycling.

As far as your RNA smearing is concerned, i think this is likely to be a different problem, such as the quality of your starting material or degradation of the RNA (thermal, or by RNases). Posting more information about your RNA sample prep and RT set up (priming method etc) would be helpful for troubleshooting.

Good luck!

We have this problem every once in a while. It's usually a sign that someone forgot to use the tube frame/rack supplied with the cycler. When loading individual tubes into the BioRAD C1000 (or other cyclers), it's best to use a tube frame/rack to reinforce the tubes and ensure the lid exerts even pressure. Another option is to put an empty tube in each corner of the block. The catalog number for your cycler should be either 1849000 or 1849001. That should keep your tubes from getting squished and probably improve your results.

The protocol looks fine to me.

My thoughts are in line with Kevin and Tania above.

The problem could be related to incompatible tubes used to run the PCR. Regarding smearing problem, I would run appropriate positive and negative controls to see if your reagents are working fine.

Good Luck.

It could be the tubes as others have said, but I also noticed you specified 40 ul sample volume in your method, while you said you have 20 ul in your post. This will likely influence the quality of your pcr reaction/product since the cooling/heating times will be slightly off.

Ana Crnkovic Hi, I hope you are fine

The volume i am using is 20ul, but the image i added here has 40ul, the was another reaction, i didn't shared the image of program with 20ul. But, another thing i found yesterday, that the tightening of lid was causing the compression of my tubes. so i again ran reaction with less compression but nothing appeared. can i used RNAses if there is contamination of RNA with my DNA samples. will this make any change to my results or reaction. Because RNA contamination can also lead to smear/tail formation is gel results. Looking forward to experts opinion. Rezwana Assad Kevin Cavallin

Yes, you should use RNase A for RNA removal during DNA extraction.

or

Try different template dilutions like: 1:10, 1:100 or even 1:1000 of template DNA prior to its use in PCR. This way you will be using very less concentration of DNA as well as RNA or other inhibitors. I guess that should solve the smearing problem and you will get good results as well.

Place your tubes in the centre of PCR and in their immediate wells place empty PCR tubes encircling your samples on all sides. This solves the problem of samples getting evaporated.

Rezwana Assad Is it possible that due to more concentration of DNA smear formation can occur. Recently, I used 2ul DNA in total 20ul vol. Still issue exists. Can there be a reason that the quality of my primers be compromised. Because, i am using them for last 4 years so there is possibility that the quality might be deteriorated due to thawing and freezing.

Yes, there is possibility that high DNA concentration cause smear. Even i faced the same issue with my samples. Then i tried diluting my template and that worked well for me. You just take 1ul of template and add 999ul nuclease free water (1:1000 dilution), then You can use 2ul template of this diluted sample (Don't dilute you whole template). You can even try different dilutions that i mentioned in earlier comments.

See, primers start degrading after you construct them by using nuclease free water. You just make working solutions in aliquots and avoid frequent freeze thaws. For checking whether primer are working or not, run a few some positive control rxns along with your samples.

I agree with dr Kevin Cavallin, and there is change in annealing temperature between picture 2 in pcr program and picture 3 in the annealing temperature was 56 but in picture 2 was 61.7

Over tightening of PCR caps has caused the deformation of PCR tubes, it is not melted. To reduce the smear formation it is better to reduce DNA concentration.