Ladder-effect seen in my pcr images!

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novice2
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Ladder-effect seen in my pcr images!

Dear All
I have started getting this ladder-like artifact in my images. I have attached 3 images that show this problem. I was wondering if any of you has seen it before and if so what is causing it. Also how can I get rid of it.
All this started very suddenly. I have been attempting to optimize this pcr assay for a few months now and it was working well. My primers are specific for my target and the yield is good.
Please can anybody help!

Ivan Delgado
Ivan Delgado's picture
 

 
Hi novice2,
Are these ladders visible both in the gel as well as your gel documentation system? I ask this because I have seen a similar effect that was due to the optics of the gel documentation system and not the PCR itself. I doubt it is the case here, but I bring it up just in case. The way you solve a gel documentation problem is to clean the inside of the chamber. Sometimes stuff gets in there that causes all sorts of imaging effects. 
If this is a PCR effect, the most likely culprit is contamination. The bands that you are getting are clearly very well defined, which suggests that you have contaminating templates in your PCR reaction. My gut feeling is that somehow you got some ladder DNA into your assay that happens to be amplified by your PCR assay. Your best bet if this is the case is to get new reagents (including your water) and run the PCR assay again. Make sure to take special caution not to cross contaminate any of your reagents. 
Good luck
 

Jason King
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This looks interesting. I

This looks interesting. I think it has something to do with mis-priming. Are your primers unusually long? What is the nature of the template DNA that you are using (cDNA? and if so, how produced?) It does look like multimerization of primer-dimer. ie. The lanes that have the strongest laddering have a very strong primer-dimer band at the bottom. It is strange that you don't get the problem in all of your samples though, so there must be some influence on the process by the template nucleic acids.
Are you using a different PCR machine now (ie. is the annealing temperature still the same as it was before you started getting the ladders?)
 
Could it be that you get the long ladders WITHOUT high molecular weight products when you have no template that the primers can anneal to (so they anneal to themselves) and ladders WITH high mol. weight stuff when you have genomic DNA contamination? (assuming that this is an RT-PCR). In the case where you get the expected band, this would be because the primers had some specific cDNA to (preferentially) anneal to.

novice2
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Hi parvoman

Hi parvoman
Thanks for the input. This is a standard PCR using genomic DNA. It was working perfectly ok for a whole year and this is the first time this ladder has appeared.

My primers are 22mer and 25-mer long; GC content is 45.5% and 40% respectively. The PCR machine is still the same. The Tm of these primers is 58oC and 59oC and since these ladders bagan to appear last week I have tried all annealing temperatures (from 57-66) and they appear at all these temperatures. As I say this was my best PCR!.

To address the contamination problem, I have used the same reagents (buffer, water, dNTPs) to amplify other genes using other primers and used the same machines, even the same positions on the hotplate and all those others were ok.

Any ideas??

Rajiv-Genetics
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What kind of gene are you

What kind of gene are you trying to amplify, and from what (cells, treated cells, tissues)? did you run a negative control (with no template added)? If yes what did you see in that reaction tube?
 

novice2
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Rajiv-Genetics

Rajiv-Genetics
I'm amplifying a mitochondrial gene using genomic DNA extracted from tissue using the Qiagin DNeasy tissue kit. I did run a negative and even the negative control (water) had this ladder effect

Rajiv-Genetics
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mitochondrial gene means you

mitochondrial gene means you are amplifying the gene present on the mitochondrial genome? Am i correct?
Also the gel figures indicate that some samples do not show the ladder effect while some do when you set up the pcr reactions? Is that correct?
Are you setting up master mixes for all the samples shown in a gel?
do the reactions of one master mix show variation i.,e. some show ladder effect and other dont when all of them were set up from the same master mix?
 

Rajiv-Genetics
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do the samples of each gel

do the samples of each gel vary by any means? what abt the lanes.. coud you indicate what the lanes represent. Is there any difference in samples of gel 1 -3. 

Jason King
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Did you say that your "water"

Did you say that your "water" control also gave the ladder? Does this control contain everything except the genomic DNA sample?
 If the water control has no genomic DNA then the bands are due to primer-dimer. You could run the water control with each primer on its own to see whether one of the primers is the cause. Maybe you just need to re-order the primers and perhaps freeze them down as aliquots to reduce the number of freeze-thaw cycles they get.
It looks like you get the ladder when there is no real template for the primers to anneal to. You could try titrating down the amount of genomic template you use - You'd need to start with a DNA sample that gives you the expected band then do 10-fold dilutions of the template in PCR reactions and see whether the single band becomes fainter and fainter before becomming a ladder.

novice2
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Hi Parvoman

Hi Parvoman
Yes the ladder is present even in my control (my control is usually water or nothing i.e. just an aliquot of mastermix to which I either add water or leave out the water). Good idea I will try running the control as you suggested with each primer to see which one is resposible for this ladder. I really think as you suggested the other day that all this is caused by multimerization of the primers. I usually aliqout my primers as soon as I reconstitute them and then freeze them down as aliquots which I thaw out once and use.
I began optimizing this PCR in May 2008 and since that date to April 2009 I have not even once seen these ladders. Now from April 2009 to mid-June I hadn't used these primers and then went back to the pcr again and that's when it all began to happen. So from that I think that it's a technical problem with the primers (multimerization). I have ordered in new primers and hope that because they are fresh primers they won't form this ladder.
I did 2 experiments yesterday: in one I heated my F&R primers at 95% for 10minutes and then placed them on ice and used them in my mastermix. In the second expt I used DMSO (1ul in 50ul rxt). The PCR with the heated primers gave me much lesser ladder (i.e. ladder apperaed in one lane as compared to 6 in the DMSO PCR). Also my negative (water) with the heated primers showed a very faint ladder at the bottom of the lane as compared to the DMSO negative where the ladder was very prominent.
I will post the results using my fresh primers meanwhile please let me know of any more ideas
Many many thanks for all your help

Jason King
Jason King's picture
I hope it works out with the

I hope it works out with the new primers. The only other thing I can think of is that certain wells in your PCR block are not getting up to 95 degrees as they should. Could there be dirt/dust etc in some of the wells? I think I'd run some samples on a different PCR machine to rule this out.
 
Good Luck!

novice2
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Hello again Parvoman

Hello again Parvoman

Since the last posting, I have ordered in new HPLC primers and the result was still the same. I then did what you suggested and used each primer on its own to see which one is resposible for the ladder formation. Although no ladder formed with either one, primer dimers/multimers were produced by the forward primer which would suggest that it may be the cause of the problem. I have attached an image for both F&R reaction. Do you think if I design a new forward I may get rid of the problem??.

I really am devastated-- this was my best PCR which was working really well for a whole year and then suddenly THIS. Also what I can't understand is that when I first designed these primers I checked them for primer dimer & hairpin formation and they were fine. Even the quality control sheets that are provided by the primer synthesis companies say that these primers don't form dimers.

Also, if I design a new forward and it works how can I be sure that a few months down the line this won't happen again?.

Awaiting your kind response.

Novice2

Collicat
Collicat's picture
 Hi,

 Hi,
I see that time has passed and am just wondering if you ever figured this out.  I have had the same thing happen but not to this extent.   With time it just went away as mysteriously as it came.

homa
homa's picture
Hi novice2

Hi novice2
I have the same problem as yours and i can't find the solution. could you solve this problem?
how can I get rid of it?
please help me in this problem

homa
homa's picture
please help me

please help me
who knows about my problem?
I should optimize my test as soon as possible.

Biju
Biju's picture
Hi

Hi
You should reduce the template DNA  for PCR , if the PCR ahs been optimized before and stopped working now. This could happen if the quality of DNA isolated now is superior compared to before.
Biju Joseph

Helena Stokes
Helena Stokes's picture
Same problem!

Hi there,I'm aware this post is from several years ago, but I'm just wondering if you had any advice on how to solve this problem? I have a similar issue, which suddenly occurred in a PCR which used to work perfectly. There is a ladder forming in many samples, as well as in the positive control and the NTC. I have tested to see whether it is contamination, having used completely new primers, reagents and nuclease free water- but I am still getting the same problem. Please let me know if you have any advice! Many thanks

Ivan Delgado
Ivan Delgado's picture
Ladder amplification in all samples

Hi Helena,If you are getting multiple bands amplifying, a "ladder", in all your samples including NTC then very likely you have some contamination or your primers are self-priming. If it is contamination then discarding all your reagents, including water and DNA, and using all new reagents should solve the problem. If it is the primers then re-designing the assay will solve the problem.Hope this helps.