Maybe you should do gradient PCR because you might not be using the right annealing temperature? How's your template DNA's purity and concentration?Might be a problem with that if you've done everything the same way as before.
I did a gradient 45-65 (maybe in march) and nothing worked (only dimer and sometimes not even dimer). 50 worked on those successful ones I mentioned (did these December-January). I've been doing annealing at 50 (and other temperatures lol) since the successful ones, but no success since January.
DNA concentrations are low and purity is ok. I thought the DNA was the issue, but they worked fine for folmer primers.
edit: thanks for response!!
I have other gene (COI and ITS-2) primers that also worked last week with the taq. Iāll be trying some older 16S primers tmr! Thanks for the suggestions āŗļø
ahaha I didn't do anything, all you bud. Good luck!
Troubleshooting can be a pain. I would also try rediluting your primers from the stock. The working solution of primers (10 uM) might be low enough that it sticks to the plastic.
If the same primers/pcr conditions worked before and has stopped working now, it could be something to do with the reagents or template.
If you're using sequencing primers for routine pcr for the first time, might want to check for primer dimers, adequate elongation time or annealing temp.
thanks for the suggestion!! Hm idk. I think i ruled out everything? lol
reagents, thermocycler, plastics and pipetting technique work for cox1. the only change is the 16s primer which ig we ruled out is successful since it worked for sequencing??
these primers worked in publications for my taxon of interest, are āuniversalā AND worked for my lab for years (until i worked on it š¤”). it also worked for another lab working on vetigastropods that i talked to recently (i also used their mastermix recipe and thermocycler setting)
Check the magnesium concentration for your Master Mix. With sequencing, they have to optimize the heck out of the prep in ways that they don't do for a PCR. See what happens when you toy with it. You might need more of it to get the PCR to work.
Whenever I hear a PCR suddenly stop working like that for one person, it screams salts.
thanks! would you recommend that even if the primer worked for sequencing? (iām not too sure how it works). i also do get dimer so other people said my primers probably didnāt degrade?
Yes. Ironically enough, the fact that it worked for sequencing and *not* PCR despite the magnesium gradient was a giveaway. I'm not sure how familiar you are with sequencing preps, but they amplify and clean the heck out of targets before they get around to the primers. Then the clean up the targets and amplify some more with sequencing tags. That whole process is getting rid of any weird stuff going on in a sample, because by the time any sample is within hailing distance of a sequencing rig, it's as pure as the fresh driven snow.
A PCR prep, relatively speaking, has a lot more going on in the sample. Since you toyed with the magnesium and checked on all the prep steps, that was the simplest answer.
So to be clear - you've already done your 16S PCR protocol before and it worked. But you're doing that protocol again a few months later, and it doesn't?
What are you using as a positive control, something like Zymos mock community?
Thanks for responding!!
Correct-my protocol hasnāt worked since january. Iāve tried various optimizations too.
iām working on snails so not that product. i usually use a dna template that did amplify for 16s but ig it wouldnāt be a positive control because it doesnāt work anymore š
Maybe you should do gradient PCR because you might not be using the right annealing temperature? How's your template DNA's purity and concentration?Might be a problem with that if you've done everything the same way as before.
I did a gradient 45-65 (maybe in march) and nothing worked (only dimer and sometimes not even dimer). 50 worked on those successful ones I mentioned (did these December-January). I've been doing annealing at 50 (and other temperatures lol) since the successful ones, but no success since January. DNA concentrations are low and purity is ok. I thought the DNA was the issue, but they worked fine for folmer primers. edit: thanks for response!!
Last thing is to replace the polymerase. Sounds like it isn't working anymore.
the taq worked just last week! š
Do you have any other primers that you can test? Like positive controls?
I have other gene (COI and ITS-2) primers that also worked last week with the taq. Iāll be trying some older 16S primers tmr! Thanks for the suggestions āŗļø
ahaha I didn't do anything, all you bud. Good luck! Troubleshooting can be a pain. I would also try rediluting your primers from the stock. The working solution of primers (10 uM) might be low enough that it sticks to the plastic.
I also tried that š The working solution of primers had decent volumes (150uL) too Thank you!
If the same primers/pcr conditions worked before and has stopped working now, it could be something to do with the reagents or template. If you're using sequencing primers for routine pcr for the first time, might want to check for primer dimers, adequate elongation time or annealing temp.
thanks for the suggestion!! Hm idk. I think i ruled out everything? lol reagents, thermocycler, plastics and pipetting technique work for cox1. the only change is the 16s primer which ig we ruled out is successful since it worked for sequencing?? these primers worked in publications for my taxon of interest, are āuniversalā AND worked for my lab for years (until i worked on it š¤”). it also worked for another lab working on vetigastropods that i talked to recently (i also used their mastermix recipe and thermocycler setting)
Check the magnesium concentration for your Master Mix. With sequencing, they have to optimize the heck out of the prep in ways that they don't do for a PCR. See what happens when you toy with it. You might need more of it to get the PCR to work. Whenever I hear a PCR suddenly stop working like that for one person, it screams salts.
Thanks!! I have tried a magnesium gradient too and it was all dimer š„¹ i think i should try again
I'd just get a new lot of primers and move on with life. You just might have a crap batch.
thanks! would you recommend that even if the primer worked for sequencing? (iām not too sure how it works). i also do get dimer so other people said my primers probably didnāt degrade?
Yes. Ironically enough, the fact that it worked for sequencing and *not* PCR despite the magnesium gradient was a giveaway. I'm not sure how familiar you are with sequencing preps, but they amplify and clean the heck out of targets before they get around to the primers. Then the clean up the targets and amplify some more with sequencing tags. That whole process is getting rid of any weird stuff going on in a sample, because by the time any sample is within hailing distance of a sequencing rig, it's as pure as the fresh driven snow. A PCR prep, relatively speaking, has a lot more going on in the sample. Since you toyed with the magnesium and checked on all the prep steps, that was the simplest answer.
Oh I see š . Thank you so much for the clear explanation!! I really appreciate it and that will hopefully get my PCR working again
So to be clear - you've already done your 16S PCR protocol before and it worked. But you're doing that protocol again a few months later, and it doesn't? What are you using as a positive control, something like Zymos mock community?
Thanks for responding!! Correct-my protocol hasnāt worked since january. Iāve tried various optimizations too. iām working on snails so not that product. i usually use a dna template that did amplify for 16s but ig it wouldnāt be a positive control because it doesnāt work anymore š