Odd Gel Run following PCR

The following is my 1% PCR gel run.  

As you can see the products have strangely run all the way until the end.  The products also look to be further than the dye looks to be on the gel itself.  I'm relatively new to PCR and not completely sure how to trouble shoot this.  The first row is a 1 kb ladder and the third row is a control.  The expected product should have had a product length of 611.  I ran the PCR at 4 annealing temperatures 5 C below the Tm.  Does anyone know where I should start in determining what may have gone wrong with my PCR/gel run?

Based on the information you provided, most likely what you are seeing in your gel is primer dimers at the bottom of the gel.

Before you start all sorts of experiment to troubleshoot your PCR, here are some suggestions: 

1. Run a PCR you know works. Get primers from someone that are known to work all the time. Then use your PCR reagents, and your DNA, and see if it works. 

2. If #1 above works, then you may want to re-design your primers (since the problem is likely the primers you are using). My rule of thumb is to test at least 2, and if you can 3, sets of primers and see which one works best. Even the best primer design program is not perfect, so instead of spending weeks troubleshooting a pair of primers that may never work, spend a couple of days running 2 or 3 sets of primers. One of them is bound to work. 

Good luck

Thanks so much.  Yesterday I actually ran a different PCR based on the fact that you guys mentioned primer dimerization.  If anybody was curious the following was my previous PCR protocol for a particularly GC rich region before I ran another PCR.

Mastermix Ingredients (vol ul)

TAQ (.3)

10x Buffer (5)

DMSO (5)

MgCl2 (4)

dNTP + 7-deaza dGTP (5)

Forward Primer (2.5)

Reverse Primer (2.5)

dH20 (24.7)

Primer Prep.

1) Took the amount of primer nMoles, multiplied it by 10, and added that amount in nanopure water to the original amount of dry primer stock to achieve a 100 pM stock.

2) Took 0 ul of that primer and added it to a new tube, to which I would add 450 ul to achieve a 1:10 dilution and a final 10 um final stock.

I then ran my PCR on a temp. gradient from 51-57 because I estimated my annealing temp. to be about 5 degrees below my Tm of 60. 

I also blasted my primers again and they seem to match up very well.  They also had been used in other experiments before as well so I can't imagine them being faulty by design, although I do have another purchased primer pair that I could try out if I give up on these.
  The one factor regarding my factors which I have a concern about is the dilution of them from the dry stock.  I didn't quite understand the math but my mentor told me I should achieve a final stock of 10 um.  She then told me to use the aforementioned protocol to achieve that molarity.  I personally don't understand the math myself. 

However, I did notice that another paper ran the papers at a higher temp.  I then decided to run the same protocol with the same primers with a different temperature gradient from 63-71.  I did achieve a band within the 3rd well at least but as you can see there is still a ridiculous amount of primer dimerization.  I've been trying to troubleshoot this PCR but at this point I personally believe it has to do with my primer solution.  I've speced my DNA samples and they look fine so I don't believe it could be the DNA itself. 

I apologize.  For my primer prep. it should say "Took 50 ul of that primer"

Here are my comments about the latest information you provided: 

1. Providing the volumes of the PCR reagents helps little if we do not know what the concentrations of your solutions are. For example you say that you are using 4 ul of MgCl2. If your final concentration of MgCl2 is less than 1 mM, or higher than 4 mM, then this could be causing problems. 

2. Not being sure what concentration of primers you are using is a problem. Your primers should be at 10 uM, which I believe they are based on what you said. If that is the case, then your final primer concentration is 0.5 uM, which is fine. Still, I am not convinced that your primers were resuspended correctly. I strongly recommend you contact the company that synthesized the primers to make sure you resuspended them correctly, and next time simply order the primers to be shipped to you already resuspended as 100 uM stocks. 

3. You use annealing temperatures of 51 to 57 degrees and of 63 to 71 degrees. You may not have noticed but you missed temperatures between 57 and 63 degrees, arguably some of the most ideal annealing temperatures for PCR. Next time I recommend running a gradient PCR from 54 to 70 degrees. 

Thanks. I'll post that below.

Stock Concentration

DMSO (100%)
10x Bufer (10x)
MgCl2 (25 mM)
dNTP + deanzaGTP (2mM each)
Forward (10 uM)
Reverse (10 uM)
TAQ (5 units/uL) 

In addition my PCR settings are below:

Aenaturation
Temp: 95
Time: 20 sec

Annealing
Temp:63-71
Time: 1 min

Extension
Temp: 72
Time:1

Based on the last information you provided, I have one additional comment: 

You did not mention a 95oC denaturation step before you start PCR cycling. Some Taq enzymes require an activation step that typically goes for 3 to 5 minutes at 94oC or 95oC. If your Taq requires activation then the fact that you do not have an activation step would explain why the PCR is not working. 

I apologize for the typo but in my previous post I wrote the following:

Aenaturation
Temp: 95
Time: 20 sec


I meant to write Denaturation.  However I noticed you mentioned a much longer Denaturation step.  Do you think 20 secs is too short?  I think my next troubleshooting step may be adding less primer solution to my mastermix.  From 25 ul to 10-15 ul from my "10 uM" stock solution.   I'm not sure if that is drastic enough a drop to correct my PCR reaction. 

I am uncertain about my resuspension steps as well, however I don't believe my mentor will work buying pre-suspended stock solution into my troubleshooting steps. 

Twenty seconds is a little on the short side for a denaturion step, but that is not what I was talking about. Before you start your PCR cycling, some Taq enzymes require an activation step. My recommendation is to read through the manual of your Taq and make sure your Taq is not one of those Taqs that require an activation step of 2 to 5 minutes at 94oC before they can start working. 

 Thanks Ivan.  I used TaKaRa   EX Taq which as my mentor told me is not a hot start TAQ and does not require activation.  I did go ahead and previously ran another optimization run using a  a primer gradient though (from .5-1.5 ul/well.  Each well containing 49 ul of Mastermix and 1 ul of DNA.

The primer gradient I set up previously turned out as follows.  

Well 1: 5 ul 1 KB plus ladder
Wells 2-3: .5 ul primer
Well 4: NC w/ .5
Wells 5-6: .75 ul primer
Well 7: NC w/ .75
Wells 8-9: 1.25 ul primer
Well 10: NC w/ 1.25
Wells 11-12: 1.5 ul primer
Well 12: NC w/ 1.5

In summary, 42 cycles, 95 C, 30 sec, 55, 30 sec, 63.1 30 sec, 72 C, 1 min

I actually don't know why the 55 30 second period precedes the ideal temperature I calculated and why I shouldn't just run the PCR annealing temp at 63.1 for 1 min.  My mentor has always set the PCR reaction up with the 55 30 sec annealing period so I left it that way. I will have to ask her about that the next time I speak with her.  One problem I did notice are the presence of multiple bands (which shouldn't happen in my PCR reaction) and some smearing.  Could this be because 42 cycles is too high? or is this more indicative of having to do a dilution series you think? 

Here are my comments based on the latest information you provided: 

1. When you estimate the Tm of a primer, that is all it is: an estimate. A primer may have a calculated Tm of 56oC and work best when the annealing temperature is 60oC. 

2. The gel image you posted shows that your primers are not amplifying your PCR fragment of interest. This could be due to a number of reasons. One could be that your annealing temperature is too low. When the annealing temperature is too low the primers bind to multiple sites and this amplify multiple PCR fragments. Another reason is that the design of your primers is not good. If the primers are too small, or the DNA sequence is not specific to a given region and instead detects multiple DNA regions, you will have the kind of results you obtained. 

In short, your PCR are workin in that you are getting PCR fragments amplified. The problem you are having is that your primers are not specific, or your annealing temperature is not high enough. 

 Hmm.  I wasn't sure if the issue was annealing temp so earlier I ran a temperature gradient from about 63-71.  I noticed I was most successful around 63 so I don't believe inc. annealing temp. will help me.  I previously blasted my primers and they were 100% homologous for the area I sought to amplify.  Is there a way I can determine the efficacy of my primers and whether they may be binding other places?

I also believe that it is amplifying the area of target interest because there is a bright band at around 611 which is where I believe there is product.  Other lanes have a bright product around 500 I believe because of  the difference in genotype, the area in question has a 48 bp VNTR.  

Sure, when you BLAST your primers you should get a whole list of DNA sequences that are homologous to your primers. If those homologous sequences are quite similar then your primers may be binding to multiple regions in your DNA.