13 replies to this topic

[EszterEmma]

Please help!

I am running PCR to test for pathogens in DNA extracted from avian RBC. Here is my problem: the reaction will periodically stop working for no apparent reason. Each time I encounter this I troubleshoot and eventually it just begins working again. Sometimes it's after I use new dNTPs, sometimes when I use fresh control DNA, sometimes I am changing several things at once so I don't know why it starts working. Invariably, however, every 5-7 reactions my positive control disappears again. Sometimes it even gets clever and gives a faint band and the time i run the reaction no band at all...I am at my wits' end trying to solve this; it's really holding up my work!


My project leader thinks that something is wrong with the tube strips and that I should be using individual tubes instead. My tube strips are designed for PCR, has anyone had success with individual tubes vs. strips?

Also, the lab protocol here is to make the master mix without water, but add water to each individual tube to dilute the DNA. Is it possible that the enzymes are too concentrated and are inhibiting each other in the master mix?

Here's my setup (per tube):

10X AmpliTaq Gold buffer = 2.5ul
MgCl2 (comes with buffer) = 2.0ul
BSA (10mg/ml) = 2.0ul
dNTP (10mM) = 2.0ul
Primer 1 (10uM) = 1.5ul
Primer 2 (10uM) = 1.5ul
AmpliTaq (5U) = 0.1ul
No water in master mix

I use 11.6 master mix in 25ul total reaction (DNA diluted in water 13.4ul)
I use generally use between 75-150ng DNA per reaction (some samples can have very low parasitemia so I tend to go heavy on the DNA)

I'm leaving out some details because i want to keep it concise, but based on this info does anyone out there have any suggestions??

Much appreciated!

[bob1]

Have you tried setting up your control reaction multiple times using the same conditions to see if it is some sort of systematic error, or something else?

If you are using a multichannel, it could be that one of the channels is pipetting incorrectly - hence the repeated error.  Also, but highly unlikely is a dead well or two in the PCR machine, especially if it is an old machine.

There could be a problem with your DNA extractions, most likely carry over of some sort of inhibitor - usually this will clear up if you do a dilution series (usu 100-1000x dilutions) on the DNA, but given your low levels of starting parasite DNA this may not be feasible to do effectively

I have used individual tubes, strip tubes and plates all with no problems for PCR;  The only problem that is likely is if the tubes have something in them.  I use tubes that are DNA, RNA and DNAse/RNAse free straght from the bag, DON'T autoclave them,this only adds stuff - similarly for tips.

[David C H]

Your final concentration of dNTP is 800uM (0.8mM), is that correct? If so, that concentration is way too high and some reactions are going to fail. It is recommended to have the dNTP concentration in the 100-400uM range. 250uM is commonly used. If you go above 400uM, some reactions will work perfectly and some will fail. I don't know for sure why this is -- it may be because dNTP degrade (even new dNTP aren't 100%) and the bad dNTP get stuck in and inactivate taq. If you have to use that much dNTP, ultrapure dNTP may help. Also, aliquot small volumes of dNTP to limit damage from freeze/thaw.

Your primer concentrations may be 3X-5X too high. Okay for some things (like degenerate PCR), but not necessary for most common applications.

Edited by David C H, 26 October 2012 - 09:57 AM.

[bob1]

High dNTPs can be compensated for by increasing the Mg2+ concentration in the reaction if necessary.  Depending on the concentration of the Mg in the 10x buffer and the added Mg you may have too much in the reaction.  I see an optimisation coming on...

[EszterEmma]

Thanks for all the input!

I've tried repeating the rxns and still get bad results (except, of course, when it randomly starts working again), I don't use a multi-channel pipette, and I just checked the calibration on my pipettes and they're accurate.

I'm relieve to hear that other people have not seen a difference with individual tubes vs. strips, sometimes I have to test 40-50 samples at a time and that many tiny little tubes is so bothersome!

This protocol was handed to me when I join the lab so I had not considered the amount of each reagent, but I will try to decrease the dNTP concentration and see if that works. If too much dNTP will make the rxn sometimes work, sometimes fail then my PCR would fit that pattern...

One more question to throw out there: My control DNA comes from two sources - extracted DNA from 2007 that has been stored in (I hope) TE buffer or fresh extraction that I've performed on saponified whole blood stored at -70C from 1999. If there's DNA degradation going on, would that account for the back-and-forth results I'm seeing? Or, would degraded DNA just not ever give positive results?

Thanks!

Eszter

[EszterEmma]

Also, my 10X buffer contains no MgCl2 do you think that the MgCl2 at 2mM final concentration in my mix is too high? I really hope I don't need to start changing around several things at once...

[phage434]

You should definitely be starting with a master mix, so there is absolutely no need to optimize anything except your program and the primer sequences.

[David C H]

EszterEmma, on 30 October 2012 - 01:57 PM, said:

Also, my 10X buffer contains no MgCl2 do you think that the MgCl2 at 2mM final concentration in my mix is too high? I really hope I don't need to start changing around several things at once...

2mM MgCl2 should be fine.

[EszterEmma]

As I explained, I do use a master mix. However, instead of adding water to the master mix, I add it to each individual tube and then add master mix to each tube.

I tried lowering the dNTP concentration to 200uM - success!! Thank you so much for the suggestion!

[Trof]

EszterEmma, on 31 October 2012 - 03:52 PM, said:

As I explained, I do use a master mix. However, instead of adding water to the master mix, I add it to each individual tube and then add master mix to each tube.

Mastermix is not only the bunch of things you mix together and pipett into wells, but also it means a complete solution (premixed) you buy from a company, so you don't need to mix or change anything.

I think phage thought the other one, something like AmpliTaq Gold PCR Master Mix (same enzyme you use) or my personal favorite HotStarTaq Master Mix Kit. You don't need to (and can't actually) opimize that, since it works on almost anythig.

[EszterEmma]

Ah, I see. I've heard of pre-mixed master mixes but have never used one myself - I think my project leader would not be too happy with me asking for commercial mastermix when we have oodles of PCR reagents in the lab...so I suppose I might have to just troubleshoot until i can get my hands on the pre-made stuff

[phage434]

If you can't use a commercial master mix, I'd suggest making large batches of your master mix.  Then the variation will be in your sample, the program, the tubes, or the cycler.  Where does your water come from?

[k_undertoe]

Previous users gave good tips, are you adhering to all the 'rules' for a good PCR? e.g don't freeze-thaw aliquots of primers, dNTPs, DNA template etc. multiple times?  Also, using different primer concentration aliquots (for ex. I aliquot 10micromolar, 20 and 50, and sometimes the 10's will go off) can sometimes affect the reaction.  If you store in TE, that can affect downstream efficiency- do you always add the same amount of starting template to your reactions, or do you 'dilute' out?  The presence of inhibitors from that source may contribute if so.

[Trof]

Hmm, rules.. I use mastermix that is in the fridge all the time, I only aliquot DNA if it's in large quantities and used very often, only have one stock concentration of primers and one "working" (the working one usualy in a fridge if being used regulary, if it stops working I make new ones, if it still doesn't, I order new primers, happend once, the primers have just had seventh birthday), I store everything in 10mM tris pH 8 only and if the DNA concentration is within certain range I always add the same amount of template,1 ul.
So rules.. well..
(this of course apply to classic PCR only because I basicaly only care if it amplifies fine or not, and in vast majority it does)