PCR Troubles! Please Help!

[wishiwasfoosin]

Ok, here's an interesting dilemma we've been having in our lab lately, and none of us can come up with a valid reason behind it, so for those of you that have any ideas as to what's going wrong, PLEASE help us out here!

This is how the PCR used to look when all things were running correctly (the lower bands are Internal Controls, the higher bands are positive reactions)

And this is how our PCR has been looking for the last few weeks :

So this has been quite baffling for us. We have tried new primers, polymerases, templates, and buffers, but keep getting this same result. If anybody has any ideas or solutions, we're all ears! Thanks for your help, and for making SDN rock!

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[Auraraptor]

Ok, here's an interesting dilemma we've been having in our lab lately, and none of us can come up with a valid reason behind it, so for those of you that have any ideas as to what's going wrong, PLEASE help us out here!

This is how the PCR used to look when all things were running correctly (the lower bands are Internal Controls, the higher bands are positive reactions)

And this is how our PCR has been looking for the last few weeks :

So this has been quite baffling for us. We have tried new primers, polymerases, templates, and buffers, but keep getting this same result. If anybody has any ideas or solutions, we're all ears! Thanks for your help, and for making SDN rock!

Did you try a different gel %?

 

[wishiwasfoosin]

Yeah, tried a 1.5% agarose gel, we usually use a 2%, it didn't change anything.

 

[Doctor&Geek]

What haven't you changed?

Water, micropipettes (use barrier tips), decrease primer & template concentrations.

 

[wishiwasfoosin]

We only use PCR grade materials, including the water and barrier tips (Eppendorf & FinnTip), I'll have to check on the template & primer concentrations, but do you think too much of them would cause that kind of effect? I ran a gel with dilutions (straight, 1:2, 1:10, 1:100) of my amplification products, and this is what i got (bottom half of gel):

It just appears to die off, as opposed to migrate! ArrrgggghhH! Molecular is half science, half voodoo!

 

[j-weezy]

Is the PCR the actual experiment or are you using PCR to confirm results (ie. are there other possible experimental pitfalls besides PCR conditions)? Are you using the same thermocycler? I used to get spastic results using Vent polymerase - it's a high fidelity polymerase but has low processivity (is that the word?) so it falls off the template rather easily. If you just want to look for band size, go for Taq (lower fidelity but super high processivity)....if it doesn't work using Taq then there are larger issues. If you need your PCR product to have high fidelity and high processivity (good if you need to know or use the sequence) use Phusion from NEB. Have you tried using controls in the thermocycler - a PCR reaction you know should work? Or do you know for a fact that the thermocycler is working just fine? If everything is working fine, then maybe try a gradient for annealing temperature. sigh..... PCR is both the blessing and the curse of modern molecular biology

after just reading the pre-allo board, i realize nothing i said is useful

 

[Doctor&Geek]

I just talked with a colleague of mine down the hall - I remember seeing something like this before where the PCR products look like they're "stuck" in the well. He replaced the polymerase & also the DNA loading dye and it seemed to fix his problem. Failing that try to get another lab's PCR reagents and rerun, or if you're really good friends, get them to do the PCR for you.

 

[gbwillner]

We only use PCR grade materials, including the water and barrier tips (Eppendorf & FinnTip), I'll have to check on the template & primer concentrations, but do you think too much of them would cause that kind of effect? I ran a gel with dilutions (straight, 1:2, 1:10, 1:100) of my amplification products, and this is what i got (bottom half of gel):

It just appears to die off, as opposed to migrate! ArrrgggghhH! Molecular is half science, half voodoo!

Dudes,
Maybe you should look into why SOME of your samples seem to work OK- INCLUDING the ladder, while others do NOT.

I agree though- it definitely looks like a gel problem and not a PCR problem. It does not look like the gel is polymerizing properly and you are getting inconsistent matrix patterns.

QUESTION: how do you prepare the gel? Perhaps you are pouring it too hot/cold? what kind of agarose are you using? How is it cooling down? How do you load the samples? How long is it taking you to finish loading?

 

[solitude]

I have had this same problem before, and it, strangely, only struck in some of the lanes. It turned out that the gel and loading dye had been diluted with TAE, but that the buffer actually in the gel-box was TBE. It's unclear if this could be the case for you, because you haven't mentioned what buffer you're using, if it is consistent, whether you have made up new gels, etc.

 

[j-weezy]

I have had this same problem before, and it, strangely, only struck in some of the lanes. It turned out that the gel and loading dye had been diluted with TAE, but that the buffer actually in the gel-box was TBE. It's unclear if this could be the case for you, because you haven't mentioned what buffer you're using, if it is consistent, whether you have made up new gels, etc.

good thinking. I always write a note on the gel box saying what buffer is in there to avoid problems for myself or others.

 

[Hohenheim]

I've experienced something like this twice. The first time, the problem was the cycler. The second time, it was a buffer problem.

 

[gbwillner]

I've experienced something like this twice. The first time, the problem was the cycler. The second time, it was a buffer problem.

One more thing- I did experience something like this once... It turned out that the Gel box was leaking. This can also happen if you run the gel too quickly (too high voltage melts the gel) or too slowly (the ETBr diffuses, leaving the "smear" look).

Good luck!

 

[wishiwasfoosin]

Is the PCR the actual experiment or are you using PCR to confirm results (ie. are there other possible experimental pitfalls besides PCR conditions)? Are you using the same thermocycler? Have you tried using controls in the thermocycler - a PCR reaction you know should work? sigh..... PCR is both the blessing and the curse of modern molecular biology

after just reading the pre-allo board, i realize nothing i said is useful

The test is a clinical one, and is used to correlate with other resutls, so i'd say that's the big pitfall of it! Using different thermocyclers, and have tried running various controls with limited success (sometimes yes, sometimes no), and PCR is definitely a blessing/curse! And you definitely said some useful things, I posted again in the scientist board for a reason!

I just talked with a colleague of mine down the hall - I remember seeing something like this before where the PCR products look like they're "stuck" in the well. He replaced the polymerase & also the DNA loading dye and it seemed to fix his problem. Failing that try to get another lab's PCR reagents and rerun, or if you're really good friends, get them to do the PCR for you.

Problem is I can run old rxns from back in the "good old days" using same gel formula and same loading dye and it migrates perfectly. Got 2 other folk to run it for me, no dice.

QUESTION: how do you prepare the gel? Perhaps you are pouring it too hot/cold? what kind of agarose are you using? How is it cooling down? How do you load the samples? How long is it taking you to finish loading?

make a 2% soln of agarose(sigma, molecular grade) and boric acid buffer, microwave 2 mins, cool(cold water on the outside of the erlenmeyer, pour, let harden >30mins. Loading time for 50 wells is between 10-20 mins.

I have had this same problem before, and it, strangely, only struck in some of the lanes. It turned out that the gel and loading dye had been diluted with TAE, but that the buffer actually in the gel-box was TBE. It's unclear if this could be the case for you, because you haven't mentioned what buffer you're using, if it is consistent, whether you have made up new gels, etc.

We're only using boric acid buffer, a 1X concentration for both the gel and the electrophoresis chamber, with Ethidium bromide in both.



Thanks for the help and advice from everyone, and i'll be sure to keep ya posted!

 

[gbwillner]

The test is a clinical one, and is used to correlate with other resutls, so i'd say that's the big pitfall of it! Using different thermocyclers, and have tried running various controls with limited success (sometimes yes, sometimes no), and PCR is definitely a blessing/curse! And you definitely said some useful things, I posted again in the scientist board for a reason!


Problem is I can run old rxns from back in the "good old days" using same gel formula and same loading dye and it migrates perfectly. Got 2 other folk to run it for me, no dice.


make a 2% soln of agarose(sigma, molecular grade) and boric acid buffer, microwave 2 mins, cool(cold water on the outside of the erlenmeyer, pour, let harden >30mins. Loading time for 50 wells is between 10-20 mins.


We're only using boric acid buffer, a 1X concentration for both the gel and the electrophoresis chamber, with Ethidium bromide in both.



Thanks for the help and advice from everyone, and i'll be sure to keep ya posted!

Quick recommendations:

1- limit time you rinse the erlenmyer flask with cold water- the gel should be ~60 degrees when you pour it.
2- make fresh buffer, but better- switch to TBE. Make FRESH.
3- run fewer samples (just a handful) until you fix the problem- you will waste time and resources until you find the problem.
4- use all new reagents- throw out old stuff.

If these don't work- get a gun.

G

 

[solitude]

Quick recommendations:

1- limit time you rinse the erlenmyer flask with cold water- the gel should be ~60 degrees when you pour it.
2- make fresh buffer, but better- switch to TBE. Make FRESH.
3- run fewer samples (just a handful) until you fix the problem- you will waste time and resources until you find the problem.
4- use all new reagents- throw out old stuff.

If these don't work- get a gun.

G

Good recommendations--I agree with all. I've never used boric acid buffer before so I can't comment on that, but when in doubt, just throw everything out and start anew with reagents that have worked in the past. I really don't think this is a PCR program, but rather an electrophoresis one. Another suggestion would be to try running the gel in a different lab down the hall, and if it works, just start buying their reagents for use in your lab.

 

[j-weezy]

The test is a clinical one, and is used to correlate with other resutls, so i'd say that's the big pitfall of it! Using different thermocyclers, and have tried running various controls with limited success (sometimes yes, sometimes no), and PCR is definitely a blessing/curse! And you definitely said some useful things, I posted again in the scientist board for a reason!


Problem is I can run old rxns from back in the "good old days" using same gel formula and same loading dye and it migrates perfectly. Got 2 other folk to run it for me, no dice.

Maybe I'm an idiot but is it possible that there's something different with your samples? Have you seen what your sample (DNA) concentration is (by Nanodrop or spec...)?

It appears that the problem is NOT with your gel as you can run old reactions just fine. You say your PCR controls only occasionally work? Even in separate thermocyclers? Are other people's PCRs coming out ok? It could still be a thermocycler issue (although I doubt it). What are your samples stored in? Have you started adding glycerol to any samples? Are your tubes DNAse free? How are you ordering your primers (I know IDT gives you options)?

 

[solitude]

Maybe I'm an idiot but is it possible that there's something different with your samples? Have you seen what your sample (DNA) concentration is (by Nanodrop or spec...)?

It appears that the problem is NOT with your gel as you can run old reactions just fine. You say your PCR controls only occasionally work? Even in separate thermocyclers? Are other people's PCRs coming out ok? It could still be a thermocycler issue (although I doubt it). What are your samples stored in? Have you started adding glycerol to any samples? Are your tubes DNAse free? How are you ordering your primers (I know IDT gives you options)?

But even when a PCR completely fails and you get no product whatsoever, the loading dye runs down the gel, and when you expose the gel you can see primer-dimers, dNTPs and all that other junk at the bottom of each lane. This makes me suspicious that it's a PCR problem, especially given that, when exposed with EtBr, there seem to be DNA products, they're just stuck at the top.

 

[gbwillner]

But even when a PCR completely fails and you get no product whatsoever, the loading dye runs down the gel, and when you expose the gel you can see primer-dimers, dNTPs and all that other junk at the bottom of each lane. This makes me suspicious that it's a PCR problem, especially given that, when exposed with EtBr, there seem to be DNA products, they're just stuck at the top.

Yup... maybe SUB justforgot to turn on the power....

Or the gel box is busted/the wires are dirty and only intermittent.

 

[surgwannabe]

genomic dna? it looks like the quality of your dna is not as good (degraded?) as the old samples. OD these samples. try the pcr rxns again by making a master pcr mix for both of the old/new samples. run them at the same time using the same reagents.

 

[solitude]

Yup... maybe SUB justforgot to turn on the power....

Or the gel box is busted/the wires are dirty and only intermittent.

good points

 

[MrMunkily]

When I was trying to diagnose a gel running problem like this appears to be, I ran my reaction products (it wasn't a PCR but it's probably applicable here too) over a purification column.
The gel purification kit we used had a seperate buffer for quick & dirty cleanup of enzymatic reactions. You might want to give this a shot. I've never seen sample get "stuck" in the well, but it couldn't hurt to make sure that it's just DNA that goes in.

 

[GvL]

Protein contamination can cause your DNA to remain in the wells.

Have you noticed any differences in your preparation of your new sample DNA before the PCR?

Perhaps you are not purifying your sample DNA correctly, and somehow either left protein in your DNA, or introduced some contaminating protein?

To rule out protein contamination, you might try to purify your new PCR product using ethanol purification/precipitation.

 

[wishiwasfoosin]

If these don't work- get a gun.

It would be more like the scene from office space where they steal the printer, except it would be my thermocyclers!

Maybe I'm an idiot but is it possible that there's something different with your samples? Have you seen what your sample (DNA) concentration is (by Nanodrop or spec...)?

You say your PCR controls only occasionally work? Even in separate thermocyclers? Are other people's PCRs coming out ok? It could still be a thermocycler issue (although I doubt it). What are your samples stored in? Have you started adding glycerol to any samples? Are your tubes DNAse free? How are you ordering your primers (I know IDT gives you options)?

Not to sound too green, but we've never checked out DNA concentration, and I'm unfamiliar with NanoDrop. I do have an old spec that i've never used though. My controls work most of the time, but there have been times when they don't replicate at all. There are no other people who use the instruments, so that doesnt apply here. The samples are stored in a buffered soln (tween, tris-Hcl, hcl, edta, and NaOH) with no glycerol, and all tubes are dnase and rnase free. I order my primers through invitrogen, i don't think there are options, i just give them my sequence and they synthesize it.

Yup... maybe SUB justforgot to turn on the power....

Or the gel box is busted/the wires are dirty and only intermittent.

Checked the box, all is good. I'm 99% sure this isn't an electrophorectic issue since i can take old amplification product and it migrates perfectly.

To rule out protein contamination, you might try to purify your new PCR product using ethanol purification/precipitation.

Ya, i've looked into it but have held off until now because i've been going from least labor intensive fixes to most labor intensive fixes . I guess i'll run a EtOH precipitation on mon.

UPDATE: I ran 4 experiments on friday, using a new inhouse(we made up the buffer) mix with internal control, inhouse without IC, invitrogen mix with IC, and invitrogen without IC. I'll post the pics tomorrow, but all the mixes without IC seem to run fine (although that makes all my negatives invisible) and those with IC are all smeared and have less robust positive amplification. This is maddening since we just had brand new IC synthesized. Maybe we need to look at a new kind of IC. Any suggestions?

 

[gbwillner]

Not to sound too green, but we've never checked out DNA concentration, and I'm unfamiliar with NanoDrop.

Anyone else hear alarm bells ringing????? I'm not sure what you're testing for, but you want to make sure to load equal amounts of DNA for all your samples. This will also test the DNA quality.

UPDATE: I ran 4 experiments on friday, using a new inhouse(we made up the buffer) mix with internal control, inhouse without IC, invitrogen mix with IC, and invitrogen without IC. I'll post the pics tomorrow, but all the mixes without IC seem to run fine (although that makes all my negatives invisible) and those with IC are all smeared and have less robust positive amplification. This is maddening since we just had brand new IC synthesized. Maybe we need to look at a new kind of IC. Any suggestions?

I never mix primers together in one reaction. BTW I really don't understand what you are doing in the described experiment. What is this "internal control (IC?) you mention? PLease provide more detailed accounts for better suggestions.

 

[j-weezy]

Anyone else hear alarm bells ringing????? I'm not sure what you're testing for, but you want to make sure to load equal amounts of DNA for all your samples. This will also test the DNA quality.

I never mix primers together in one reaction. BTW I really don't understand what you are doing in the described experiment. What is this "internal control (IC?) you mention? PLease provide more detailed accounts for better suggestions.

Yes, there appears to be a slight lack of QC with regards to sample quality. Always check your OD! I've lost so many weeks on experiments that didn't work because I was lazy and didn't check the OD reading of my sample. I agree with gbwillner, it's always better to set up reactions based on amounts (know how many micrograms or nanograms you're adding), not just volume.


I wonder if the OP includes a known template with the same (or different but unique) primers to act as an internal control for each PCR reaction. Although it sounds strange, i can't think of what else this IC could be.

 

[wishiwasfoosin]

What is this "internal control (IC?) you mention? PLease provide more detailed accounts for better suggestions.

The internal control is human genomic DNA that we have replicate simply to ensure that amplification is occurring, and that no inhibitors to PCR are present. The tested is a 2 round nested PCR to detect the presence of dermatophytes in nail tissue. On friday's experiment, each reaction was separate, i ran 25 reactions (controls, pt negatives, pt positives) each of inhouse with IC, inhouse without IC, etc.

Always check your OD! I agree with gbwillner, it's always better to set up reactions based on amounts (know how many micrograms or nanograms you're adding), not just volume.

I wonder if the OP includes a known template with the same (or different but unique) primers to act as an internal control for each PCR reaction. Although it sounds strange, i can't think of what else this IC could be.

I'm on the clinical end of things, and the samples i receive for testing are toenails. We digest them in NaOH and then freeze thaw. We then take a small volume of that sample, add it to tween, and incubate at 94C fof 10mins(to remove inhibitors). Then we add our buffer soln to bring the pH down and pipette out 1uL to add to our primer/nucleotide/template mix. I'm not sure where we would OD the test at. Your analysis of our IC is correct, it is a template with different primers that we add to the mix. If there is no fungal amplification (the fungal bands work fine, it's the IC that appears to be the issue) then we look for IC amplification so we can safely report as negative. This is also a qualitative test, so would OD or Nanospec still come into play?

 

[j-weezy]

Your analysis of our IC is correct, it is a template with different primers that we add to the mix. If there is no fungal amplification (the fungal bands work fine, it's the IC that appears to be the issue) then we look for IC amplification so we can safely report as negative. This is also a qualitative test, so would OD or Nanospec still come into play?

Ok so I'm slowly coming to understand the whole process.

Do you know the what the concentration is of your control template? PCR can amplify tiny amounts of DNA but it can't amplify from nothing. My first suggestion would be to take your template and find out what the concentration is. I may be beating a dead horse but have you run a sample of your IC template (unamplified)? I can't remember if you said so in this thread or the other thread and I'm lazy. So you've figured out the thermocycler works just fine, there's no problems with the gel or associated buffers. Run a gel of your unamplified template alongside your PCRed template. If neither runs, it's a template problem (either protein contamination, degraded, or just nothing left). If the unamplified template runs but the PCR amplified template doesn't, there's something wrong with your PCR conditions. Maybe someone already suggested this - I can't remember. But now that I understand the actual problem, that's my only advice.