30 replies to this topic

[lab rat]

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 [MgCl2] and see which reaction gives the best bands.

Edited by lab rat, 13 November 2009 - 10:24 AM.

[almost a doctor]

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

[lamb]

Hi tomato!

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

[tomatoNC]

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

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[lab rat]

What happens if you substitute DMSO for BSA?

[tomatoNC]

I didn't use DMSO or BSA. I had been using 1ul of BSA per tube but I stopped. I have never used DMSO, if the primer dimer problem had been more prevalent I was going to use it. What concentration should I try?

[tomatoNC]

I think I still added too much PCR product (ie shouldn't have reamped the already reamped) but at least it shows that there is no hope for set 2. Here are the initial criteria

Set 1:
1X Buffer (final conc.)
200µM dNTPs (final conc.)
0.5µM each Primer (final conc.)
2.5mM MgCl2 (final conc.)
0.2µl 1X Taq

1µl 20 ng/µl DNA per sample
total volume 25µl

Re-amplification 1:
5ul PCR product, same MM and total vol

Re-amplification 2:
2ul product from reamp 1, same MM and total vol

Two major questions-
1) Where should I go from here. Should I reamp the original PCR product again using less product?
2) Why is there still a big blurred area in the negative control (and the other lanes)?

I think I will dilute the second reamp product and run it on the gel again.

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[lab rat]

When I have a template that is difficult to amplify, I try adding DMSO to enhance the reaction. I think the optimum is usually around 5% of rxn volume, I usually add 0.25 or 0.5 ul per 50 ul reaction. (I don't worry about percentage.) You can optimize the concentration to what works best for you.  

The BSA or DMSO are both adjuvants to help make the DNA more accessible to the enzyme. DMSO specifically helps with strand separation in GC-rich regions.

[Adrian K]

Hi tomato, I tried to analyze your primer (P7-43DF3/R1 ), it seems that your primers have high primer dimers...

CACGGGATATG  TT    RT  TG ATAAG  CATGT
____________|___|__|___|__|___|__|||
____________C CA    CT  AT TTCAA  GGACACCATTTCTG

As suggested by lab rat and others, 5% of the DMSO can be substitute your BSA.
Also, try run a gradient temp PCR range from 50 to 65.
I suspect your annealing temperature should be around 56~58C5 (althought published temperature might be different).

Here are my humble suggestion:
Try to Keep your MgCl2 final conc at 1.8 or 2.0, DMSO 5%(I use in all my pcr, works fine)

Also, I doubt whether your template got starch contamination... as the smearing is quite heavy.

Furthermore, For Takara Taq 10X buffer there are 2 types, One is not mgcl2 free and another is mgcl2, which could be the problem. Hope you didn't took the wrong one...

just my 2 cents.

Edited by adrian kohsf, 07 December 2009 - 03:39 PM.

[tomatoNC]

The 10X buffer does not contain MgCl

I added 5% DMSO (final conc.) to the PCR reactions today but I am still seeing primer dimers and no bands. The reaplification product is still smeary and appears to be too large to be the desired band.

Wells 1-4:
1X Buffer (final conc.)
200µM dNTPs (final conc.)
0.5µM each Primer (final conc.)
2.5mM MgCl2 (final conc.)
0.2µl 1X Taq
1µl 20 ng/µl DNA per sample
5% DMSO
total volume 25µl

Well 5: Negative control

Wells 6-8, 10:
2X buffer (final conc.)
2.5 µl of 2.5mM dNTPs
0.025µl each Primer
2.5µl of 25mM MgCl2
0.1µl Taq
1.875 µl DNA
5% DMSO

Well 9: Ladder
Well 11: Negative Control

Wells 12 and 13: reamplification of PCR product (same as Wells 1-4)

Well 14: ladder
Well 15: Negative Control

Wells 16 and 17: reamp (like wells 6-8,10)

Well 18: negative control

I think I am going to try a temperature gradient next, but shouldn't I at least have faint bands first?
Where do I go from here, I'm beginning to be afraid that my boss thinks I'm an idiot. Help!

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[lab rat]

You are not an idiot. Failure-angst is part of the PCR experience.  

Try Adrian's advice with the gradient and [MgCl]. I'll keep thinking on it, and I'll ask one of my plant science colleagues about this.

[phage434]

How much time are you willing to waste before you redesign your primers?  I can't believe that people spend weeks trying to make poor primers perform in pcr reactions.  Is your time worth so little?  A good primer pair should work without spending weeks fiddling with annealing temperatures, magnesium concentration, or template amounts or additives.

I've never been able to successfully reamplify a weak band in a pcr reaction and get a strong band.  I always get smears.  I don't even try any more.

My top suggestion:  Use a PCR premix rather than mixing all components yourself.  Avoid the temptation to fiddle or optimize.  Let the pros do that for you.  Design primers for annealing at 55C.  If they don't work, try a gradient annealing temperature.  If they don't work and it is high GC, add 5% betaine.  If they don't work and are very low GC, lower the extension temperature to 65C.  If you suspect inhibitors, dilute 10x and 100x template and retry.
This should take a maximum of two days.

Next, throw those primers out and redesign them.

Favorite enzyme mix: Invitrogen PCR Supermix High Fidelity
Don't bounce around and try a dozen -- learn how to make one of them work well for you.  Phusion would be another good choice.

[Adrian K]

Dear TomatoNC,

What happen to your ladder? why it seems very smearrrr? is it self-made? Or is it something have to do with your gel?

No harm trying, try lower down your [MgCl] concentration, as I suggest 1.8mM to 2.0mM will do. I think 1.5mM will work as well. For My personal experience, if more than 2.0mM my PCR will become haywired.

As phage434 said, dilute your template. There is less need to quantitate the DNA, just try to dilute it will do 10x or 100x.

Regarding DMSO, try to do a same set without DMSO. Maybe in this rare case, without DMSO will be better for you??? Who knows....

We will do our very best to help you be labeled you as genius by your boss....
Good  luck.

[tomatoNC]

They just ordered new primers before I got here, so I don't know if redesigning primers is an option.
I am going to try the gradient and diluted template.

I think the ladder was smeared because it was diluted and the power source isn't great.

I appreciate all the advice! Keep it coming if you think of anything else!
Thanks