Odd gel run following PCR - (Jun/28/2012 )
I'm glad to see less non-specific binding as well. I apologize for being less specific in my wording however. I actually was running a temperature gradient. Wells 3 and 4 were run at 58 C (different controls), 6 and 7 at 59 C, 8 and 9 at 60 C, etc.
I have also quantified my template (I believe). I'v included my mastermix procedure below:
Thaw buffer dNTP, MgCl2, and primer stock solution. After thawing, vortex well
Keep TAQ Polymerase in freezer until needed.
Into a 1.5 ml microcentrifuge tube add 272 ul dH20
Add 50 ul 10x buffer
Add 40 ul MgCl2
Add 12.5 each of the F & R primers
Add 50 ul DMSO
Add 50 ul dNTP + 7-deaza dGTP stock solution
Add 3 ul TAQ Polymerase
Vortex reagents to ensure mixing of master mix.
PCR Tube Preparation
Add 49 ul Mastermix /well
Add 1 ul DNA / well
As my last step indicates, I add 1 ul of DNA/well. If I run less template tomorrow will that increase primer dimierzation though since less template will be available for binding?
So how much template do you add to each reaction then? (amount of DNA, not volume)
What is the concentration of your template stock?
princeofnam on Mon Jul 23 23:16:25 2012 said:
Do you require an initial lower temp for temperature gradient runs?
Also the answer to this question is no.
Think about how a PCR works.
First you heat the sample to a temperature that causes the double stranded template to denature and "unwind", making it accessible to primers and polymerases.
Secondly you lower the temperature such that binding of your primer to the template can occur. If you use a temperature that is too low, the specificity of this annealing is low, so you can get non-specific binding of your primer to non-target sequences.
Then, in your extension step, the polymerase adds dNTP to your primer, creating your amplicons. Each non-specific specific amplicon will now have the complementary primer sequence at the beginning of the strand- making it even easier to be amplified in the next round.
In each repeated step, you are making more and more of these non-specific products. Even if you were to then increase the annealing temperature to a higher temp after this point, you already have a great deal of non-specific product around, and these will be amplified, as well as your specific product.
That is why having an initial, lower annealing temperature is a really really bad idea.
Unfortunately I'll have to come into lab tomorrow to check my lab notebooks nano drop data. To my memory both controls had ng/ul values of around 150-180 ng/ul. So I suppose I'm adding about 150-180 ng per tube? I'll be even further sure when I check tomorrow.
Unrelated but possibly a factor. I run 1.5% gels. I mix 20 ul of pcr reaction with 4 ul of loading due and then load that into each well.
If you are adding 150ng per reaction, then what we have been saying all along (that you have too much template) is likely the problem.
What is your target DNA? Genomic? Plasmid? etc...
Most protocols recommend amounts of DNA of about 1 to 10 or so ng per reaction (sometimes less sometimes a bit more depending on what it is).
I'm assuming that as you are using 7-deaza dGTP, that your target is GC rich? So in that case some protocols will call for a bit more template (say 50ng) but even then, you have well above the required amount.
If I were you, I would chose one of the controls (or even do both) and do a dilution series. Run tubes with 1ul of your stock neat, 1/10, 1/100 and 1/1000. I am fairly confident that this will solve your problem.
Humour me, just do it
Oh and by the way, that black spot in your gel is probably a small clump of agarose that wasn't fully dissolved when you added your EtBr, so it doesn't have any in it and hence appears black. Nothing to worry about.
I spent today running a dilution series. I ended up with odd results again.
Wells 1, 2: 1 ul of ~190 ng/ul DNA
Wells 3: NC
Wells 4,5: 1/10 Dilution
Wells 6,7: 1/100 Dilution
Wells 8, 9: 1/1000 Dilution
I just wanted to point out that I noted before that I ran the 95 C denaturing step for 20 secs. I've actually been running it for 30 secs.
95 C, 5 min
40 cycles, 95 C, 30 sec, 63.1 C, 30 sec, 72 C, 1 min
72 C, 5 min
During this run I also neglected to prepare my PCR tubes on a frozen tray, instead using a room temperature tray. Part of my protocol before was to prepare the PCR Tubes on ice. I'm not sure if this may have affected my PCR Run. I also ran the gel in a larger container with more TBE. However, I still used a 75 ml 1.5% gel. Lastly, I'm using a new batch of DMSO that just came in.
The serial dilution series was completed using four initial PCR tubes.
Tube 1: 2.5 ul DNA
Tube 2: 9 ul H20
Tube 3: 9 ul H20
Tube 4: 9 ul H20
I then pipetted 1 ul of DNA from Tube 1 into Tube 2, mixed.
I then pipetted 1 ul of DNA from Tube 2 into Tube 3, mixed.
I rechecked the nanodrop data and the Control samples had around ~190 ng/ul concentration.
I then pipetted 1 ul of DNA from each tube into their respective final PCR tubes with 49 ul Mastermix. I used two controls per DNA amount as well. What strikes me as odd is that I didn't even see the usual first bands with the 1 ul amount which leads me to believe I may have messed up during my PCR tube preparation. I also am wondering whether I should return to a 1 minute annealing temperature. On the plus side, there appears to be less smearing. Or maybe I should use different primers. Both seem to match for my target DNA sequence though when I blast though. And the one I'm using now has less overall self-complimentarity.
I've been reading into other methods and materials for people trying to look at the same VNTR as I am. Ive noticed these other researchers have employed other methods such as hotstart as well as the "touchdown" PCR which you mentioned.
A few things.
It is unlikely that not setting up on ice is the cause of your problem (provided you add the enzyme to your master mix last, and as long as you work fairly quickly). I never set my PCR reactions up on ice, regardless of the polymerase I'm using, and it hasn't caused problems for me. (it would typically take about 10-15 min to set up a PCR reaction with 10 or so tubes).
Secondly, I would probably have used one of the lower annealing temps for this cycle. Your 63C bands from your gradient are quite weak so there is a good chance that this temp is a bit hit and miss for your primers. I would stick to maybe 58 or 59. I re-looked at your gradient gel and I think that besides the smearing, you aren't getting any non-specific binding at these temps so they should work well.
Thirdly, I think you are right in that given even your neat samples failed this time, that it was probably an issue with this set up.
Wow, this must be so frustrating for you!! I hope you can get to the bottom of it!
Do you know what though, I think if you have some other primers that could work on your template- you should try them. From that we'll at least be able to see if the template is ok (and not degraded etc) and get an idea of how much template to use. Good idea!
I ran the dilution series again exactly as before except I used leftover DNA from yesterday (stored in the freezer). Strangely, still no product for the usual amount of DNA (1 ul/well). I can't really figure out why this is occurring. At any rate, I've set up my PCR today to be a temp gradient again from 57-62 C. I've also run each temperature with a 1ul/well control along with a 1/10 dilution control. In addition I increased the annealing time from 30 seconds to a minute as I previously had. If this doesn't turn out well I may have to see whether my shipment of DMSO is the right kind (is that even a problem?). I also had to mix my own dNTP + 7-deaza mix for the first time, which may also confound my results. At any rate, I think I may try to use a new primer if all else fails. I really chose a tough PCR to tackle for my first PCR experiment!
I. Can't. Believe. It. I just looked at my gel comparing one run with my old batch of DMSO and another run with my new batch of DMSO (the batch I've been using for my runs since coming back from vacation. The old batch of DMSO run has bands showing up again with my new batch exhibiting little to nothing (like I've been showing on this forum for my last posts). The new batch, the batch I personally ordered is Hybri-Max grade Sigma DMSO which I was told to order from a PCR protocol I read online. The old batch I borrowed from a friend. He described his DMSO as "cell-culture" grade DMSO which as pointed out "is very good. At any rate, I'm not sure what I should do at this step. Should I order his grade of DMSO or is it possible to achieve his DMSO-grade by substituting more of my DMSO? My mentor already ordered for me 4 10ml flame sealed ampoules of the not-so-great DMSO.