Most people who answered here, like many reviewers, are functionally illiterate it seems, sorry, and missed the point that your PCR used to WORK. HEnce changing some  basic  numbers in the protocol will not help. There must be something that sneaked in in a procedure that is NOT atiucalted in ususal protocols. These may include, beyond typical batch-batch differences::

* PRIMERS:  Freeze-thaw should be fine. BUT:  if you stored  the working stock (rather low con) in small plastic tubes, they may eventually, due to repeated exposure to RT, adsorb to the tube wall. Some plastic does that more than others (use low adsorbing plastic - more expensive). Go back to your concentrated stock now and then!

* If you use the old fashioned mineral oil method to cover samples: Here are  batch-batch differences!

* HOT-START procedure: do you use a chemical/biological or physical hotstart?   If you use the later, perhaps a slight change in "work habit" can affect our result. You may have primers that strongly depend on hot start. Sometimes 5 min exposure to RT between going from ice to the 95 deg C of the PCR cycler (which MUST  be preheated to 95 deg in case you do not do so already to get consistent results) can completely prevent amplification (due to pre-PCR mis-priming and undesired elongation at the wrong site by a few nucleotides  rendering priers useless) 

* As some have said: Cycler may be another variable not captured by protocol.   Not only precision of temp but the rate of ramping up to desired temp may affect result in cases of non-robust primer design

Do you see unused primers on gel in your failed PCR reactions? If yes, they are fine, and if not, may be they are degraded.

I'm guessing you prepared a working dilution of your primers; you can try preparing a new dilution and check if that works. What do you dilute your primers in?

Ok, first of all, are you running a positive control in your PCR. If the answer is yes, and you obtain a product with your posirive control, then maybe the problem is with your template DNA; if there is no product from your positive control, and you have changed all the reactives of your PCR kit, maybe you could have a problem with the Thermocycler... Have you run a different PCR in it? If you didn`t put a positive control and you have the chance to run one, do it... Anyway check the thermocycler or try in a different one... If there is no problem with it and you still have not PCR product, have you saved the one you obtained the first time you use your new primers? Do you have any chance to sequence it? Time ago I have a terrible experience with primers that formed concatemers with the same size than the expected PCR product!

Good luck!

Theresa, you do not have to trouble yourself. Definately, something is wrong along the line but you have to be systematic in finding out the problem. First, what temperature did you keep the primers? It will be fine at -20 to -80C, If kept at that temperature then the primer is ok. Next, is to ensure like Monica said to include a positive control and if the positive control does yield the expected product size, then you can suspect the reagents. That is if the positive control is properly kept at the right temperature (usually +4C before reconstitution and -80C after reconstitution). This way you will be able to tell if it is the template that has degraded. By the way, did you do a one step reaction or cDNA? Usually it is advisable to make cDNA as it is more stable, if you have to come back to the same template. Next, is to check your cycling conditions and be sure the conditions are same as was earlier used. Of primary interest is the annealing temperature, be sure it is exactly the same as before. Off course, you can run the template and the primer on a 2% agarose to see.  

Thanks for all your answers!

The primers were diltued in sterile water and are stored at -20 °C. I made a new working dilution, which did not work either.  I use cDNA as template and i also tried different stocks.

The thermocycler works fine for other PCRs. So this should not be the problem.

I run serveral PCRs with a temperatur gradient. Very surprisingly in the first test PCR, which worked quite nicely, the optimal annealing temperature was alot higher than the expected T and above 72 °C. An Annealing temperatur of 79°C yielded one precise band of the expected length 2 kB. We then also tried a two Step PCR, but this did not work.

On the gel i can see the unused primers. Intrestingly, the (reverse) primer seems to form secondary structure (second band). This second band i could not observe, when i used 3 mM MgCl2 instead of 2 mM MgCl2.

Theresa, I also had the same problem like u.

I think u can do the following things

1) Check the water quality, used for primer dilution (I think u used Mili Q water)

2) If u designs the primers and used it first time then u has to re-conform the primers bioinformatically, their binding site, product site etc (though you have a definite product previously, but it happened sometimes...)

3) If u sure about primer design then the manufacturerization of the primer is not done well. Sometimes some salt can hamper the PCR reaction. In my case I ultimately found that the new set of primer (purchased again, HPLC grade) working well. If u using desalted primer u can use HPLC grade primer.

So I think, brought one set of primer again and check it. I can say from my own experience, this can save lot of time as because I lost mine for the same reason.

Theresa,

our experience is that the quality of water is limiting. Sometimes happen, when you are preparing water for PCR by yourself, that water is not such a pure as you expect or that your water is no more sterile and it is contaminated.

Do other PCR reactions, with the newly diluted (other) primers, work properly? If it is the problem of water, problems should be everywhere.

primers for another PCR were diluted in the same water at the same day. And these primers work just fine.

Dear Theresa,

The last time somehting like that happens to me turned out to be due to the mother solution of dNTP being not anymore dNTP because the microtube has been left too long at room temperature.

Change this solution for a brand new one ... it might very well be the reason ?

The point here is that you have to not worry too much: once a PCR worked fine, it will always work fine AGAIN ... sooner or latter.

Best regards,

Prof. Philippe Urban

Repeated freezing and thawing of primers may not advisable. Sometimes if you are dissolving in normal water, there is a chance for degradation of primers. Hence, i suggest you to make a fresh dilution of original stock and aliquot into appropriate volumes which can able to avoid the repeated freeze-thawing. 

Also you need to avoid vortexing the PCR template. 

you have to check the annealing temperature , there might be some error .

Also, you need to add enough DNA template (30-50 ng)

enough primers 10-40 pMole

Check , the sequence of the template and the primer for self-biding , hairpins or loops.

Use proteinase K  to remove proteins from DNA

you can check  the primers on gel but you need high concentration of agarose (2-3%) or Acrylamide and you need to use thehigh concentration  primer stock .

you can make a fresh dilution of original stock and aliquot into appropriate volumes as  Sharath Chandra

you can use a different PCR machine or check that it is working properly by trying another PCR reaction.

Also, you need to check the Taq enzyme (you can try another batch or manufacturer, and to check the MgCl concentration,

Finally, you can use another source of dH20

Viability of your template as stated above is a potential problem. Contamination with DNase is another. If I have an issue I mix up fresh dNTP's and use fresh Taq, also I've seen many be careless with hygiene in their work area and I would rather err on the side of caution and be very, very, clean. Remember your working with very small quantities prior to amplification so small changes can have great impact.

Depending on your mix you may need to be extra precise with pipetting, small amounts require careful attention to ensure proper deposition and quantities. One last thing, what are your primer's characteristics? Some are more stable than others.

My friend had the same problem. After having to endure with so many steps for troubleshooting, we finally found that the actual problem is that the PCR machine was broken thus providing unstable temperature during amplification (even though it seems worked well). If all the suggestions above could not solve your problem, you may need to check your machine.

In my opinion, the annealing temperature is very high. What is the melting temperature of your primers?

Hi, I had the same problem. The PCR reaction I used to do often did not work for me anymore. I tried to change all the material, I did all the technique in other lab with their kits and machine. Finally I decided to do gradient PCR to check different annealing temperatures. I am not able to explain, but the reaction was working with higher annealing temp than before - we are still using the same primers and the kit...

Maybe you have a problem with a thermal cycler. Sometimes it depends on specific position in cycler. I and my friend had a similar problem. 

Most people who answered here, like many reviewers, are functionally illiterate it seems, sorry, and missed the point that your PCR used to WORK. HEnce changing some  basic  numbers in the protocol will not help. There must be something that sneaked in in a procedure that is NOT atiucalted in ususal protocols. These may include, beyond typical batch-batch differences::

* PRIMERS:  Freeze-thaw should be fine. BUT:  if you stored  the working stock (rather low con) in small plastic tubes, they may eventually, due to repeated exposure to RT, adsorb to the tube wall. Some plastic does that more than others (use low adsorbing plastic - more expensive). Go back to your concentrated stock now and then!

* If you use the old fashioned mineral oil method to cover samples: Here are  batch-batch differences!

* HOT-START procedure: do you use a chemical/biological or physical hotstart?   If you use the later, perhaps a slight change in "work habit" can affect our result. You may have primers that strongly depend on hot start. Sometimes 5 min exposure to RT between going from ice to the 95 deg C of the PCR cycler (which MUST  be preheated to 95 deg in case you do not do so already to get consistent results) can completely prevent amplification (due to pre-PCR mis-priming and undesired elongation at the wrong site by a few nucleotides  rendering priers useless) 

* As some have said: Cycler may be another variable not captured by protocol.   Not only precision of temp but the rate of ramping up to desired temp may affect result in cases of non-robust primer design

Hi Theresa,

It seems that your primers have a tendency to self-anneal/form secondary structures, which may make your PCR difficult but it sounds like it worked fine the first time.

Depending on how your primers were handled, your PCR might work the first few times but thereafter fail; this problem can be particularly acute when your primers tend to do either of the above. 

The suggestion concerning doing a manual hot start even if you are not using a HotStart polymerase is an excellent one that will help reproducibility. I would make sure to assemble your reactions on ice as well, which also helps reproducibility.

I'd be happy to help you troubleshoot further.

I hope you have solved the problem. If not, In addition to above recommendations check your calculations (the amounts you added to PCR mix) sometimes simple calculation mistakes can be problem. Secondly, you may check  electrophoresis conditions,  run previous positive PCR products with your new PCR products. 

In my experience, primer stability is rarely, if ever, an issue in causing failed PCRs. (Remember that primers, as well as all other reagents in a PCR reaction, are expected to function more or less normally after being boiled and cooled up to 50 times.) If self or cross annealing were a problem with any primer pair, this would be expected to manifest as primer dimer that appears during the PCR reaction. Any primer-primer interactions that form during storage at cold temperature will be melted during the first denaturation step of the PCR. The introduction of any nuclease that might degrade single-stranded oligonucleotides is also generally unlikely, although that would probably be dependent on what other activities are common in the laboratory.

If you really want to run your primers on a gel, check out the attached pdf which describes how you can do that with a relatively simple agarose gel. You can make 200X Li-Boric Acid gel buffer (1 mM Li buffer described in the paper) as follows :

4.2 gram Lithium hydroxide

25 gram boric acid

water to 500 ml.

(The Brody et al. authors do point out however that this formulation is not a ''buffer" but rather a "conductive medium".)

There can be various reasons: The reagents themselves (like not maintaining proper storage condition which degrades the primes and probes and enzymes of course) and the machine and the run profile: have to be careful with the same running parameters as you have conducted before. So, it would be ideal first to check whether the reagents are properly handled, kept and used and then run the same reagents following the same parameters as previously done. Also, check the positive controls you used before, whether its compatible or not. IF all is okay, then sometimes the run program in the machine may be mistakenly used the wrong one, which may cause such troubles. You can check the electrophoresis results between the previous amplicons and later ones. You can get some clues from this in which steps it might have been mistaken: like if you see bands on the gel in later amplicons also, it indicates that there have been PCR reactions but the machine (realtime) does not show amplification due to some errors while no band at all indicates no amplification during PCR that means might be problems with your reagents as mentioned before.

Your DNA probably is degraded.

Remeasure your template concentration by Nanodrop to rule out the degradation of it and avoid repeated freeze-thaws of your primers, dNTPs. Otherwise, PCR reactions are very consistent.   

Hi Theresa.,Could you please tell me how you solved the issue finally..because iam also facing the same problem.

Hii Everyone ,

I am facing the same problem. I have verified my machine with other PCR the result is same. and when I have given my sample to other PCR again the same blank gel. The gel is only showing the ladder or the marker bands. recently I changed my set of primers and PCR MIX. And it was working good. but suddenly this nonsense is occurring. I don't have a clue what is going on. each time I am using new DNA sample. So What could be the problem.please help me. I have attached the file that have the results of last few days before and after the problem.

Dear all I also have the same problem. the primers that work very good before is not working now. I go through the suggestions given above, and changed dNTPs, have checked the qualtiy of DNA used (its good), however there is absent of band. its gradient PCR. I hope to get some informations on my problems. Thank you.

Hello  Theresa Schacht, please let us know If Did you solve the trouble with your PCR? Was the thermocylcer? regards, Ma. Johanna.

i am also suffering the same problem.For  a long period of timeThermocycler not were used in lab. After 2 years i did pcr in it and i get product.but after that i never get the product. Is it due to temperature variation:?

Dear all, I am having a similar challenge of inconsistent results with exactly the same set of reagents except templates. I sometimes even have my positive control that previously amplified failing to amplify again with same reagents, thermocycler and thermal conditions. Kindly assist. Thanks

 Dr Sui Huang can you please explain what you meant by the abbreviation "RT" when you are explaining "Sometimes 5 min exposure to RT between going from ice to the 95 deg C of the PCR

"Sometimes 5 min exposure to RT between going from ice to the 95 deg C of the PCR cycler (which MUST  be preheated to 95 deg in case you do not do so already to get consistent results)"

Thanks

dear Theresa

i have this problem too.

i do two reaction for any of my DNA, but i see the band for one micro tube but cant see the band for another one..please help me

Hi Theresa,

I am facing the same problem. I have repeated everything, used positive controls, did fresh dilutions of primers, bought new primers, bought new Taq, prepared fresh DNA samples with high purity, did serial dilutions...nothing is working. Yet it worked perfectly before. Three times. Trouble shooting for the past two weeks has been unsuccessful.

I'm facing the same problem, can you please help me to figure out what the problem is? if you have solved it?

Hello Theresa

You can find many references on attachment 1

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwisreGJ5s_WAhWrHpoKHTxGDP8QFggsMAE&url=http%3A%2F%2Fwww.modernmuzzleloader.com%2Fforum%2Fviewtopic.php%3Ft%3D11079&usg=AOvVaw3ShS7VqgdnjGfx4n8dsBKN

2

https://www.famic.go.jp/ffis/oie/obj/GMO.pdf

3

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=0ahUKEwisreGJ5s_WAhWrHpoKHTxGDP8QFgg6MAQ&url=https%3A%2F%2Fwww.thehighroad.org%2Findex.php%3Fthreads%2Fwolf-primers-what-do-you-think.476227%2F&usg=AOvVaw3p0UhvCexLCaWFFMqb95xr

4

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwisreGJ5s_WAhWrHpoKHTxGDP8QFgg-MAU&url=https%3A%2F%2Fmonitoriadegenetica.files.wordpress.com%2F2014%2F10%2Fprenatal-diagnosis-of-hemoglobin-disorders-present-and-future-ok.pdf&usg=AOvVaw2Qg_JA-9sqV8RK_XpWwz9O

5

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwiU6YmP9c_WAhWqBZoKHdA8CVUQFgg8MAU&url=http%3A%2F%2Fwww.sichelzellkrankheit.de%2FTraeger-Synodinos.pdf&usg=AOvVaw0AeC2I8UVYOrSvp7ixxdDN

6

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&uact=8&ved=0ahUKEwiU6YmP9c_WAhWqBZoKHdA8CVUQFghFMAY&url=https%3A%2F%2Fclinicalepigeneticsjournal.biomedcentral.com%2Farticles%2F10.1186%2Fs13148-015-0072-6&usg=AOvVaw30BK5Wi_bHxmg0KhUebZox

7

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&cad=rja&uact=8&ved=0ahUKEwiU6YmP9c_WAhWqBZoKHdA8CVUQFghLMAc&url=https%3A%2F%2Fissuu.com%2Fmarvinsunderground%2Fdocs%2Fcereal_genomics_methods_and_protoco&usg=AOvVaw3pYkQ3DqGNznMb-9q7fOmg

8

https://orca-mwe.cf.ac.uk/84501/1/ejhg2014131a.pdf

9

https://quizlet.com/175685046/bioe-206-entire-collection-flash-cards/

10

https://patents.google.com/patent/CN103233068A/en

11

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=0ahUKEwiRuKbX9c_WAhUFYpoKHcmUAAs4ChAWCDEwAg&url=https%3A%2F%2Fclinicalepigeneticsjournal.biomedcentral.com%2Farticles%2F10.1186%2Fs13148-015-0072-6&usg=AOvVaw30BK5Wi_bHxmg0KhUebZox

12

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=0ahUKEwiRuKbX9c_WAhUFYpoKHcmUAAs4ChAWCDEwAg&url=https%3A%2F%2Fclinicalepigeneticsjournal.biomedcentral.com%2Farticles%2F10.1186%2Fs13148-015-0072-6&usg=AOvVaw30BK5Wi_bHxmg0KhUebZox

13

https://www.highveld.com/pcr/pcr-troubleshooting.html

14

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0ahUKEwiJrJqG9s_WAhUCQJoKHXgKDvMQFggyMAI&url=https%3A%2F%2Fwww.researchgate.net%2Fpost%2FCan_someone_help_me_with_my_primer_degradation_query&usg=AOvVaw0Uwc3fds1CvWZPtUG0Nbxh

15

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=0ahUKEwiJrJqG9s_WAhUCQJoKHXgKDvMQFgg1MAM&url=https%3A%2F%2Fwww.researchgate.net%2Fpost%2FProblem_with_primers_for_long_range_PCR_that_work_for_several_reactions_and_then_stop_working_Can_anyone_help_with_this&usg=AOvVaw0urxDMLrSlaOmRY32m_djz

16

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwiJrJqG9s_WAhUCQJoKHXgKDvMQFgg_MAU&url=https%3A%2F%2Fwww.researchgate.net%2Fpost%2FDoes_anyone_have_problems_with_degraded_DNA_and_primer_dimers_after_isolation_of_DNA_from_buccal_swabs_with_QIAGEN_DNeasy_Blood_and_Tissue_Kit_250&usg=AOvVaw23MwxCDcRSM5L71MdWlMEB

17

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&uact=8&ved=0ahUKEwiJrJqG9s_WAhUCQJoKHXgKDvMQFghFMAY&url=http%3A%2F%2Fwww.sigmaaldrich.com%2Ftechnical-documents%2Farticles%2Fbiology%2Ftroubleshooting.html&usg=AOvVaw2MpMd5y8r602thwCCF5SEo

18

https://www.protocol-online.org/biology-forums/posts/3542more5.html

19

https://genepool.bio.ed.ac.uk/sanger/Sanger_troubleshooting_guide_v1.pdf

20

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0ahUKEwiJrJqG9s_WAhUCQJoKHXgKDvMQFghdMAk&url=http%3A%2F%2Fwww.nature.com%2Fnprot%2Fjournal%2Fv1%2Fn3%2Ffig_tab%2Fnprot.2006.247_T2.html&usg=AOvVaw0WbTakA-sqmfO_-ALTsoPe

Hi Theresa,

I am facing the same problem. I have repeated everything, bought new primers, did fresh dilutions of primers, bought new Taq, prepared fresh DNA samples with high purity, ..nothing is working. Yet it worked perfectly once I used the new primers first time. Trouble shooting for the past two weeks has been unsuccessful.

i am facing the same problem

Your primers got dephopshorylated. Oxygen free radicals can do that. Need new ones or phosphorylate the old ones.

Dilute the DNA in TE buffer and try with increased amount of template DNA 200-250ng, it may work.

i m also facing the same problem.getting inconsistent results, at times getting gel images blank bt only ladder is visible on gel.i want to know is it the primer issue?

Check your primers!

My PCR worked once, then it stopped working when I repeated it 3 times. I decided to use other primers which were approxiametly 10 bp longer then my old primers, and it worked perfectly again!

I guess the one time it worked with the old primers was a lucky hit.

Hi Yannis Gounaris

About your answer, I would like to say that de-phosphorylation of the primers will affect the 5' of the primers but the 3' will be ok so the PCR failure couldn't be as a cause of primer dephosphorilation. On the other hand, 3' OH removal on Carbon 3 could be a cause.

Most probably your dNTPs are old or you are using too much target DNA (more than 50 nmg per 50 μl reaction).

I am facing same problem. I tried to change many things like cdna concentration annealing temperature even polymerase. Still nothing is working.

Hello All Researchers

I am also facing the same problem

I have to amplify HVR-1 of malarial patients infected with Plasmodium falciparum

For which i have to perform two PCR

Primary and Nested PCR

The reaction preparation and conditions are given as follows:

Taq Buffer: 2.5 microlitre

MgCl2: 1.5 Microlitre

Taq polymerase: 1 microlitre

dNTPs: 1.5microlitre

Primers:1 microlitre Each (F& R)

DNA: 2 microlitre

while for Nested PCR i dilute the Primary PCR products 10X and then use 2 microlitre of it.

PCR water: 14.5microlitre

Conditions:

Stage 1: Primary Denaturation: 94 degree for 5 Mins

Stage 2: Denaturation: 94 degree for 1 minute

Annealing: 55 degrees for 1 minute

Extension: 72 degrees for 2 Minutes

Stage 3: Final Extension: 72 degrees for 7 Mins

No. of Cycles: 35

Looking Forward for Valuable suggestions.

i am facing a similar problem. I am using EF1 and EF2 for Fusarium identification. 2 weeks ago, the PCRs worked. Now its not working. troubleshooting by changing primers, DNTP, MgCl and water is not working. a positive control and using a different gene region are working. What could be the problem.

What do you advise?

I am also facing a similar problem. using Primers LR1 and NDL22 on maize plant roots DNA. Initially I had good amplification but it is not working anymore. I have changed the taq, purified the DNA further but still not getting amplicons. what could be the problem

I am also not getting pcr result I don't know where is the prob my lab ate used same dna have positive of same bacteria result but i didn't get it

@ Humaira Are you using the same set of primers and the PCR mix which your labmate is using.

I got the same problem ! really strange.

I got the same problem, one month before I got amplification but after that, I am not getting a band. even after changing the whole kit including cDNA.

@Rohit Gupta. Same here. But again after about an year the same PCR started answering with clear bands (ITS specific primer). But when I sequenced the results were not acceptable (peaks were irregular). How can this be overcome? It would be really helpful if anyone can provide any suggestions.

Swetha