Dead genotyping PCR - I can't see s*** (Dec/13/2010 )

Hi all, I should introduce myself as this is my first post. My name is Angus Wong, graduated with B.S. for 2years, working at a UCSF lab, hoping to get into graduate school soon.

So I've done PCR before, and for the longest time, I thought PCR is pretty much bulletproof, but I am wrong (apparently).

I'm new to my current lab and I've been doing a lot of genotyping, and this is what I see on my gels.
1) Just ladder
2) Faint band (KO especially, but WT too sometimes)
3) Giant smear (KO especially, rarely WT)
4) Inconsistent positive bands from gel to gel of the same sample. (KO exclusively)

For the longest time, I suspected that it's human errors on my behalf. Although this PCR has been problematic for other lab members, it's been working fine until my arrival, and I've already eliminated the possibility of bad reagents. So last week, me and a post-doc ran another PCR together. We used the same reagents, same DNA samples, same protocol, same PCR machine/program. Then I loaded everything onto the same gel.

The post-doc's PCR turned out to be GREAT, it was like Christmas lights!! Mine was complete disaster! As usual, my WT shows up, but fainter than what she was used to. My KO did not show at all.

I've done PCR before, but they were on plasmid DNA. One of the "guru" of the lab suggests it's because with plasmid DNA, my target is much bigger relative to the amount of DNA in the reaction, so it's more tolerant to minor mistake(s). Whereas in genomic PCR, because the target is so much smaller relative to the amount of DNA in the reaction, it's not as tolerant and minor mistake(s) will be amplified.

So it's coming down to pipetting and benchtop preparation. This is how I make my master-mix. (order of reagents added)
Water -> PCR Buffer -> Mg2+ -> dNTP's -> Primers -> Polymerase last
Then aliquot my master-mix into the PCR tubes
Finally add my genomic DNA.

Everything is on ice the whole time. Do you think I'm adding my TAQ too early, and by the time I get the whole thing into the machine, the TAQ is already dead, even if it's on ice sitting in the master-mix?

Thanks, I hate genotype PCR.

I understand your pain, but don't hate PCR because it is not working right now. PCR is your friend and all you need to do is simply spend more time getting to know its little tricks.

The fact that you have a post-doc that was nice enough to run the experiment side by side with you is wonderful. You now know that the problem is you (sorry, I know it sounds bad, but it is a great start).

Your problem could be the Taq, but I doubt it. Hopefully you are using a Taq that needs to be activated (heat activation, etc), which means that it does not really matter how long it is in the PCR mix before you start the PCR. Yet, if your Taq is a cheap Taq that is always active then that could be a source of your problem. If your Taq is a cheap Taq then you want to work fast and add the Taq at the last possible moment. Other than that what you are doing is all just fine.

You may not have heard of this before but we all have nucleases in our skin, which will degrade DNA upon contact. Some people have more than others, and some have a lot. If you happen to be one of those nuclease overproducers, then that could also be part of the problem. If you are not using gloves to prepare your PCR try some gloves on. That could make all the difference.

Finally, if you can convince the post-doc to run the test with you one more time (a pizza may do the trick ), I would do it again and look at how he/she performed every step (I mean EVERY) step of the process and make sure you are doing everything the same (including making sure that every time you pipette a solution you have the solution in the pipette tip. Pipetting style and process is a very important thing to master, especially when using viscous solutions like Taq that require reverse pipetting for adequate sample dispensing.

Hope this helps. Good luck!

Thanks for writing back.

Well I'm running another PCR, but this time just with our positive control and water. I had the post-doc to sit right next to as I'm preparing the master-mix. She approved of everything I do except for the Taq. Like yourself, she recommended for me to add it at the last possible moment. I followed her way and it went like this

Master-mix minus the Taq -> prepare/label the PCR tubes -> add genomic DNA to PCR tubes -> add Taq to the master-mix -> aliquot and run it.

I'll see in a few hours.

I also talked to another post-doc and she does it my way (everything into the master-mix -> aliquot to PCR tubes -> genomic DNA -> PCR Machine), and it works for her (most of the time).

I guess it's just a shock to me, I've never had PCR problems (of course I was doing it with plasmid DNA). Genomic genotype PCR is a whole other beast!

And I always wear gloves, frequent changes too.

My guess is that you didn't adequately mix in the Taq - it is quite dense relative to other components as it is suspended in a glycerol solution, such that it will sink to the bottom of the master mix tube. Some people advocate vortexing, but usually flicking the tube or 10-20 strokes of a pipette set to 1/10th of the volume of the master mix is usually enough to mix it properly.

I have never had a problem with Taq going off between adding it and getting reactions onto the machine - remember it is temperature stable to 72 degrees for several minutes so it should be able to handle a fair bit of time on ice. Having said that, it is a good idea with any enzyme to keep it in the freezer for as long as possible and only get it out for adding to the master mix/reactions so as to minimise the chance of temperature related degradation.

So I showed the post-doc my notebook and what not, she saw something strange with my dNTP mix, they were not at 10mM as the protocol has it.

The way I made dNTP's and it's always worked for me... 1:10 dilution from the 100mM stock. So I end up with 4 tubes of 1uL dA/C/G/TTP, and 9uL of water. Then I add everything into another tube so I'd have 40uL of "10mM" dNTP's. Turns out, I've been doing this wrong and I've been using super-diluted dNTP's. Every time I mixed those 4 tubes of dA/C/G/TTP together, I'm really adding more and more water to the solution.

Right now I just made a "real 10mM" dNTP's mix.

80uL total volume:
8uL dA/T/C/TTP from 100mM stock solution = 32uL
48uL water

Why 80uL? Because I just got used to making an 80uL dNTP solution.

And I guess this is why I'm still learning and these people are post-docs.

I'll update as soon as the PCR is done, but I have a good feeling about this one.

Yep, it was the dNTP's. I'm getting my bands back, nice and strong too!