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Confusing bands from PCR - not primer dimer, not product (Nov/06/2009 )

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The stock concentration of the dNTPs and Primers are 10mM. I do make the master mix myself based on what the lab technician before me did, I think I will have to make some changes. In the sample the concentrations are as follows:
dNTPs: 1.28 mM
MgCl2: .25mM
Primers: .6mM
10X: 2X


Thanks

-tomatoNC-

the final concentration of 10X should be 1X. This can mess up your reaction. I can vouch from personal experience.

Your dNPTs should be around 200 uM, your primers around 0.5 uM, and your Taq should work fine at 1 U/reaction. Mg varies; I add 1 ul of 25mM to a 25 ul reaction.

-lab rat-

lab rat on Nov 9 2009, 08:45 PM said:

the final concentration of 10X should be 1X. This can mess up your reaction. I can vouch from personal experience.

Your dNPTs should be around 200 uM, your primers around 0.5 uM, and your Taq should work fine at 1 U/reaction. Mg varies; I add 1 ul of 25mM to a 25 ul reaction.


Your primers and dNTPs concentration are way too high, I always use dNTPs at a final conc of 200uM, and primers range between 0.5-0.2uM of each primer (500-200pM). Also, your MgCl2 is too low, usually add between 2-5mM in the final mix.

I still think your annealing temperature is too low. So, exces primer, and low temperature = primer dimer. :D

-almost a doctor-

Hi Tomato,

Have you seen this publication by Barrillas? http://tgc.ifas.ufl.edu/vol58/vol58.pdf

Do a search on your primer names and you will find the article with the SOP. I think this will help you with your problems.

Regards,

lab rat

-lab rat-

Thanks for all of the advice, but I am still having problems. I ran some samples with the guidelines from the forum.
Set 1:
1X Buffer (final conc.)
200ÁM dNTPs (final conc.)
0.5ÁM each Primer (final conc.)
2.5mM MgCl2 (final conc.)

0.2Ál 1X Taq and 1Ál 20 ng/Ál DNA per sample
total volume 25Ál

And some samples based on the paper that labrat posted. I followed their procedure exactly.
Set 2:
2X buffer (final conc.)
2.5 Ál of 2.5mM dNTPs
0.025Ál each Primer
2.5Ál of 25mM MgCl2
0.1Ál Taq
1.875 Ál DNA

The conditions for both reactions were from the paper

94C 3 min
94C 30 sec
53C 1 min
72C 1 min
72C 10 min
35 cycles

Neither reaction produced any bands, but at least we got rid of the primer dimers :).
Actually, I think they may be faintly present in the four samples next to the ladder. Those samples were from set 1.
Attached Image

-tomatoNC-

Hey, that's progress. I did some reading; your organism is really interesting. :)

I'm not sure which Taq you are using, but I'm guessing that it is <5U/ul>? Have you tried setting up different reactions using increasing amounts of Taq? (say 0.1 ul-0.4ul?)

The protocol that I am currently using calls for 0.5 ul of DeepVent polymerase <2U/ul> in a 50 ul reaction. Another protocol that I have used in the past called for 0.3 ul Taq--I don't remember what concentration, but I got it from Promega--in a 20 ul reaction.

Otherwise, I would do as previously suggested: set up a series of increasing and see which reaction gives the best bands.

-lab rat-

Hi Tomato, a silly question: have you checked your template? Are you able to amplify anything from this DNA?

-almost a doctor-

Hi tomato!

What if your amplicon is just too difficult for Taq? I would try some proofreading polymerase, e.g. Pfu, Phusion or KOD.

-lamb-

The DNA polymerase is TaKaRa Taq, 5U/ul
I have varied the concentration 0.1, 0.25, 0.4
The tube I draw from just says Taq 1X, I didn't make the solution.

I have checked the DNA template, but only so far as confirming that DNA is present. What other tests should I do?

I have tried different MgCl concentrations (1mM and 2.5mM final concentrations), how high can I go?
Thanks very much

-tomatoNC-

I tried re-amplifying the pcr product from the forum suggestions and the published procedure and I got some bands. The first three lanes are based on the forum suggestions (lanes 3 and 6 are negative controls) and the second three are from the published data. I am going to re-amplify the products of this reaction but use less of the PCR product in an attempt to get clearer bands (2ul). Any other suggestions with this new data?
Thanks very much
Attached Image

-tomatoNC-
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