PCR problems - Fungal DNA (May/23/2007 )

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A little big problem....

I have confirmed the presence of my extracted DNA.
(I work on basidiomycetes), however, I haven't been able to get any positive PCRs in the last two weeks.

My reaction worked before and gave me beautiful bands where I expected them. But, it seems that something has gone wrong.

This is what goes iinto my reactions.

Primer pairs: ITS 1/LR3, ITS 1/LS1R, B2R+, Fung 001-R ( itested in several combinations)


10X PCR Buffer 3.00 (1X)
50 mM MgSO4 1.20 (2mM)
10 mM dNTP's 0.60 (0.2mM)
Primer 1 1.50 (0.25 uM)
Primer 2 1.50 (0.25 uM)

Taq Polymerase 0.12 (HiFi Taq 1 U/rxn)



The master mix (buffer, Mg, dNTPs, P1, P2, taq and water) add up to 10 uL.


I use 2 uL DNA and 18 uL of water, and then do 2, 5 and 10 of that with water to make up 20 uL.

To that 20 uL, I add the 10 uL of the master mix


Final Volume 30 ul

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My amplification settings are the following:

The settings are:

2 min at 94 c
20 s at 94 C
30 s at 52 C
60 s at 72 C X 30

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7 minutes at 72 C (final extension)

So, the biggest problem that is happening right now, is that I seem to be getting genomic bands instead of the desired segments.

I have originally thought that the issue here is DNA overloading.
I use 2uL DNA and 18 uL of water. Out of that, I take 2, 5 and 10 uL into the 30 uL reaction.

After that, I have tried higher dilutions (1:100)....but, same thing happened.

Another important thing is that positive controls (various known samples of Penicillium) are not being amplified as well. This tells me that something is wrong with the mix that I use, and not with my DNA.

My supervisor got me HiFi Taq. However, he did not supply me with a new tube of MgSO4...the one I am using is possibly several years old...maybe more than 5.

My questions are:

- Does anyone see anything that is so obviously wrong with my reactions?
- Could the problem be in the old MgSO4?
- Can I subsitiute MgSO4 with MgCl2 and use that with HiFi Taq?
- What about the HiFi buffer? Can regular PCR buffer work with HiFi Taq??

- Could the problem be in dNTPs (another questionable ingredient in my lab!!!)??

- I have tried increasing the annealing temp from 52 to 55 C and no improvement.
- If DNA overload is the case, does it make sense to increase initial denaturing time or temp??


So, many questions, so little time.
Thanks to anyone who has the patience to read this and help me out.

I know that I always use MgCl2 in my PCRs, I'm not sure what type of Taq I have though. If you're using the same primers as you were in the ones that worked a while ago they may have gone bad by now, my primers usually don't last more than 5 or so months. You might want to fiddle around with the annealing temperature more too, one time when I had a pair of primers with unusually high affinity for each other, I had to adjust the annealing temp like 10 times before it worked.

I'm kind of new to research, but those would be my first thoughts.

Get new aliquot of dNTP's. This could help.

Also check if the template DNA is fine by running it on gel.

Dilute your sample DNA 10x and 100x in TE and try the reactions using the dilutions.

When things go wrong it is usually the template.
Do you have any plasmid DNA which you can use for your PCR kit on. Something that is sure to work. Is it possible to "borrow" some dNTP, buffer etc from a friend in another lab to do test your polymerase and template.

However if you are sure it isn't the template's fault, throw everything into the bin. (leave the polymerase for last cause it is more expensive), in my experience it isn't worth the time or the effort to find the component that has gone bad or contaminated with nuclease. You win no stars here. This is why you have aliquotes, and thus able to bin anything at the tip of the hat.

"Does anyone see anything that is so obviously wrong with my reactions?"
Nope nothing obvious. Although I am not a fan of master mixes. I perfer adding each component myself

- Could the problem be in the old MgSO4?
Unlikely. Magnesium sulphate is a salt. It will never go off. Although contamination is possible. The stock of 50mM Magnesium chloride I am using was made by a former labmate 5yrs ago.

- Can I subsitiute MgSO4 with MgCl2 and use that with HiFi Taq?
You can. What is the rest of your buffer made of though? Look up the company that the Hifi Taq comes from and find out what's in their buffer.

- What about the HiFi buffer? Can regular PCR buffer work with HiFi Taq??
Unfotunately regular PCR buffer has littke meaning. There are many Taq PCR buffers in regular use. Which is your regular Taq buffer? Taq buffers
Mine is Buffer B. However, I am in the opinion that taq doesn't really care and works more or less on anything.

- Could the problem be in dNTPs (another questionable ingredient in my lab!!!)??
Anything is possible. It could be the dNTPs it could also be the primers. Or horror yet the polymerase. The fact is, the possitive control is also not working.

- I have tried increasing the annealing temp from 52 to 55 C and no improvement.
How is the negative control? Is it clean of these bands? Banding in the negative control points at contamination of master mix.

- If DNA overload is the case, does it make sense to increase initial denaturing time or temp??
Nope. It would make little difference.