28 replies to this topic

[princeofnam]

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?

[leelee]

Those aren't PCR products per se, they are primer dimer.
Your PCR has failed.

Can you start by telling us your PCR parameters (e.g. number of cycles, length of each step, temperatures used for each, which polymerase you are using, etc).

[kaveh]

As leelee mentioned, these look more like primer dimers, which are the undesired products of the PCR reactions. So, basically, there is no product on your gel, or it's too little of it that you cannot see it. Either way, this means that there is something wrong with your reaction. Depending on your samples, setup and protocol, this can be due any or a combination of these reasons:

1) You don't have much template DNA/cDNA/RNA in your reactions. > Check your sample concentration with spect
2) Your primers are not designed well. > Run a primer blast with your template on NCBI.
3) The protocol that you are running is not optimized for the template and primer sets.
4) etc..etc...etc... (Welcome to the world of PCR!)

Honestly, I would start with #1. That seems to be the culprit.

-k

[phage434]

It's almost never that there is too little template.  More often there is far too much.  Most often it is primer design, more rarely cycling conditions.

[princeofnam]

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 50 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 which I have a concern about is the dilution of primer 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.

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.

Edited by princeofnam, 01 July 2012 - 07:00 PM.

[leelee]

I think you might be adding too much primer to your reaction.

When I use taq for PCR, I typically use 0.5 to 1ul of primer (from a 10uM working solution).

[kaveh]

You have primer dimers and that shows that you have enough of the primers (or maybe mor ethan enough!).
I would increase the cycle number for few more cycles to see if the band show up.

[leelee]

How many cycles are you currently running? And for how long at each temperature?

Also, have you tried doing a dilutions series on one of your DNA templates? Say a neat, 1/100 and 1/1000?

[princeofnam]

My mentor mentioned my not having to perform a dilution series but I will mention the idea to her.  Currently I run the PCR at 40 cycles.  I will also see whether reducing the amount of primer I add to the PCR reaction will improve matters.

[leelee]

If you are already running 40 cycles (which in my opinion is too many already, I would aim for about 30), then you can cross adding more cycles off of your things to try.

It would be really helpful if you could post your PCR cycle settings, you may be doing something that is really easy to fix and might be obvious to someone one here.

As in fill out the following for us:

Denaturation
Temp:
Time:

Annealing
Temp:
Time:

Extension
Temp:
Time:

You should know what these are and why each temperature has been selected, as this will help you to trouble shoot in situations such as this.

[princeofnam]

Sure.


Aenaturation
Temp: 95
Time: 20 sec

Annealing
Temp:63-71
Time: 1 min

Extension
Temp: 72
Time:1

[princeofnam]

In addition my stock concentrations are below:


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

[princeofnam]

Hi Veteran, thanks for the tip about cycle number adjusting.  However if my band is faint shouldn't I increase my cycles from 40 to about 45ish?

[kaveh]

40 cycles are way too many already. more cycles only mess with your results further. I would use more template per reaction.

[leelee]

princeofnam, on 02 July 2012 - 08:31 AM, said:

Hi Veteran, thanks for the tip about cycle number adjusting.  However if my band is faint shouldn't I increase my cycles from 40 to about 45ish?

If a PCR isn't getting you a good product by about 30 or so cycles- then there is something wrong with some aspect of your reaction. You are better to optimise the PCR rather than simply increase the cycles, which is a quick "fix" that may not work, and doesn't address the actual problem in your reaction.

Putting your reagents through excessive cycles will diminish them, and your polymerase will start to lose activity. Also I've been told that the likelihood for errors increases.

You said you checked your DNA template by spec- so can you tell us how much DNA you add to each reaction- that should tell us whether the amount of template is the problem (highly unlikely, it is almost never the amount of template that is the issue).
I know you said you didn't want to run a dilution series with one of your templates (or you supervisor said not to) but don't forget too much template is a PCR inhibitor.

Here is what I would do:
using the template that got you the faint band in your second attempt:

1. Do a dilution series, and set up PCR tubes for neat, 1/10, 1/100 and 1/1000

2. Add master mix (using only 1ul of primer rather than 2.5ul)

3. Run the PCR with your 40 cycles (but I would probably reduce the annealing time to 15-30 sec), and I would also reduce the denaturation temperature to 94C (this is optimal for taq). You could also check the product insert for your taq to see what they recommend.

4. Use the annealing temperature that worked for your successful reaction above