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Odd Gel Run following PCR

Odd Gel Run following PCR - PCR - Polymerase Chain Reaction Forum

Odd Gel Run following PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-29-2012, 04:22 AM
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Default 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?
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  #2  
Old 06-29-2012, 05:23 AM
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Default Re: Odd Gel Run following PCR

Hi Princeofnam, it look like more of primer dimer than pcr product. If you don't mind to share your PCR contain? and also the pcr protocol.
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Old 07-02-2012, 02:56 AM
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Default Re: Odd Gel Run following PCR

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 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.
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Last edited by princeofnam; 07-02-2012 at 02:57 AM.
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Old 07-02-2012, 06:03 AM
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Default Re: Odd Gel Run following PCR

If primer dimer is not a problem with your PCR (like you can live without it) then just proceed on. There are few ways to remove it. But my brain is fried with deadline so will probably get that in later.

What is your number of cycle?
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Old 07-02-2012, 06:06 AM
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Default Re: Odd Gel Run following PCR

Btw your mentor is correct on the x10 then add that much NFW. As far as I know nowaday the company that synthesize the primer for you usually include the amount of NFW you need to add to make 100uM.

If you are doing Msc or phD, you would most DEFINITELY have to know where do you get that number. Cheers
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Old 07-02-2012, 04:30 PM
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Default Re: Odd Gel Run following PCR

Thanks butters. I ran the PCR reaction at 40 cycles. I am considering adding more to increase the presence of my band. Furthermore I am thinking about reducing the amount of primer solution I add to my mastermix from about 25 ul of a "10 um" solution to about 10-15 ul of it. I am hoping that produces less dimerization.
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Old 07-05-2012, 05:23 PM
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Default Re: Odd Gel Run following PCR

H again. I went ahead and ran another optimization run using a a primer gradient (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?
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  #8  
Old 07-06-2012, 02:55 AM
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Default Re: Odd Gel Run following PCR

55C i assume will amplify most primer. Anyhow is the size your looking for is in well 3, 6, 9, 12?
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Old 07-23-2012, 11:22 PM
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Default Re: Odd Gel Run following PCR

I'm not sure which well will have the size I'm looking for. The DNA I amplified is a polymorphic region with a 48bp VNTR. The 4R allele should be around 611 bp.

I performed another reaction with poor results as well. I made some modifications though.

) I dropped down to 40 cycles from 44
2) I ran a PCR Temp Gradient from 58-63 C
3) I took out the 55 C step.

I'm unsure about the giant black spot on my PCR. I any event, lanes 3-11 are my temperature gradient from 58-63. Lane 5 is my control. Lanes 3, 7, 9, 11 are control 1, and Lanes 4, 6, 8, and 10 are another control2 . There seems to be a consistent band for Control 1, although you can sometimes see it in Control 2. As always the bottom bands are indicative of rampant primer dimerization which I haven't figured out yet. There at least seems to be bands from non-specific binding as indicative from my previous post, although I'm not sure either if this one band I'm seeing is non-specific too. One of the bands I'm looking for is ~611, although other VNTR polymorphisms might be present in 48 bp intervals. Pretty lost as to what my next run should be. Perhaps higher annealing temperatures?
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Old 07-23-2012, 11:39 PM
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Default Re: Odd Gel Run following PCR

I also wanted to note that each well had 1.25 ul of reverse and forward primer each. I thought about reducing my primer concentration but I don't think there was much of a difference in my primer gradient run.
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