PCR product stuck in agaros wells - (Dec/05/2008 )

I ran Gel Electrophoresis on my PCR product and am finding that there is a lot of product in the wells. I do have a band of product right where I should, however it is somewhat faint.
I have read that too much template/ over amplification can case the DNA to get stuck in the wells but my template was diluted 1:100. Its possible it needs a 1:1000 dilution, but can any one explain to me *why* over amplification would cause that?
I would expect that an over amplification would give you streaking or a very large messy band at your expected length.

I also read that over drying the DNA could cause this effect as well.
I can't seem to find out how much drying is considered *over drying* nor why over drying causes it to get stuck in the wells.


I PCRed a region of my plasmid 5kb long. The Plasmid was purified using a Quiagen Midi plasmid purification kit.

If anyone could explain just what is happening under those conditions to make it remain in the wells or have any other ideas why I have DNA stuck in there I would greatly appreciate it.
Nicole

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what is the buffer you are using, type and % of gel? Did you also mix your loading dye properly?

usually over drying DNA causes it to get stuck on the tube wall and hard to dissolve in water, making it difficult for you to get any DNA template in the first place.

well, if you have too much genomic DNA template and your gel is 2%, it would make sense that the DNA wouldn't be able to get through the holes in your gel and therefore get stuck there. Overamp as well, as at say 75V for 45 minutes, only so much PCR product will be able to make it through the holes at the well into the gel, so too much product will accumulate at the well itself, unable to pass through, like a traffic jam on a highway.

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I'm using .8% agarose in T.E.A. Buffer. Loading dye was added at 1/10 vol and mixed until uniform.
Gel was run at 60V for 60min.

I'm observing primer dimers at the bottom of the gel so I no longer suspect over amplification but unfortunately don't have any other idea of what could be causing this

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What does your starting template DNA look like on a gel? If you're adding too much to the PCR reaction, that might be what you're seeing.

Can you show us an image of the gel?

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I've never uploaded images before, I hope it worked.
The 1st one is the gel of my plasmid. 1st lane is 1kb ladder, and the 6th lane is my plasmid.
The last image is the results of PCR on this plasmid. I was amplifying a 5kb region and used a temp gradient(55-65) to determine the optimal conditions.

Original concentration of plasmid, per nano-drop, was 71ng/ul.
I diluted this 1:100 and used that as my template for the PCR.

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I have the feeling that this PCR isn't working at all.

Is it possible to linearise your plasmid, cutting outside the area of interest? I believe that using a linearised plasmid template would be helpful to your PCR.

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I would agree that its not working as well as I'd like it too, But I'm getting bands in the correct area and I *think* those bands would be stronger if all the product runs.
That being said, what specifically about the gels makes you think that the PCR isn't working at all?

I didn't think of linearizing it. That might be possible.
I'm very new to all of this, so I apologize If I sound ignorant, but why would a linearized plasmid make a better template than a circular one? With the exception of a super coil, I would think that the primers would bind and extend regardless of conformation.

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Well, the images could be improved (just a matter of technique, but I can't help you there, sorry), but I reckon there's enough detail to be useful.

First of all, could you please write down your exact PCR protocol? Also, what Tms do you have for your primers, and what calculation did you use? Have they been tested for primer dimer formation and secondary structures? What primer concentration do you use? Is there anything unusual about the target DNA? High / low GC?

If you diluted the template 1:100, it's down to ~0.7 ng/ul. That is pretty low. I would try 1ul neat template or 1/10 dilution.
The MidiPrep kit is typically pretty good, so I doubt that is the source of the problem.

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[/quote]

First of all, could you please write down your exact PCR protocol? Also, what Tms do you have for your primers, and what calculation did you use? Have they been tested for primer dimer formation and secondary structures? What primer concentration do you use? Is there anything unusual about the target DNA? High / low GC?

If you diluted the template 1:100, it's down to ~0.7 ng/ul. That is pretty low. I would try 1ul neat template or 1/10 dilution.
The MidiPrep kit is typically pretty good, so I doubt that is the source of the problem.
[/quote]

10ul rxn

1ul dnpt
1ul Pfu Buffer
.5ul Pfu
1ul up Primer[10mM]
1ul down primer[10mM]
1 ul template
4.5ul ddH20

35 cycles
95 degrees C 2 min
95 30s
60 30s
72 7min
Final extension
72 14min

The melting temp I'm using is 60 degrees C, I asked the grad student I'm working with about the calculations and those weren't done. We used a temp gradient to determine the optimal temp. We are, admittedly, still working the kinks out of that. They have not been tested for primer dimer formation nor secondary structures, however, Primer dimers were assumed by the cloudy region that showed up at the base of the gel.
The target DNA is somewhat low in GC and other than being a large piece to amplify, I don't think there is anything special about it.

I'm am undergrad and this is my 1st quarter working in a lab, so I'm sorry that I seem to ignorant, but would any of this give rise to why the Product is remaining in the wells or just how to better run the PCR?
Either is fine by me, I'll take information where ever I can get it

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