28 replies to this topic

[princeofnam]

Thanks Veteran.  Before I could run a dilution series my mentor wanted me to run my PCR on a primer gradient first from .5-1.5 ul/well.

Btw, usually I add 49 ul of mastermix w/ 1 ul of DNA to each well.  The primer gradient I set up previously turned out as follows.  

Well 1: 5 ul 1 KB plus ladder
Wells 2-3: .5 ul primer
Well 4: NC w/ .5
Wells 5-6: .75 ul primer
Well 7: NC w/ .75
Wells 8-9: 1.25 ul primer
Well 10: NC w/ 1.25
Wells 11-12: 1.5 ul primer
Well 12: NC w/ 1.5

My mentor suggested I run it at more cycles contrary to your advice actually and we ran it for 42 cycles.

In summary, 42 cycles, 95 C, 30 sec, 55, 30 sec, 63.1 30 sec, 72 C, 1 min

I actually don't know why the 55 30 second period precedes the ideal temperature I calculated and why I shouldn't just run the PCR annealing temp at 63.1 for 1 min.  My mentor has always set the PCR reaction up with the 55 30 sec annealing period so I left it that way. I will have to ask her about that the next time I speak with her.  One problem I did notice are the presence of multiple bands (which shouldn't happen in my PCR reaction) and some smearing.  Could this be because the number of cycles as you mentioned is too high? or is this more indicative of having to do a dilution series you think?

[leelee]

I think that the multiple, non specific bands are due to you using a 55 annealing temperature as part of your cycle. I really don't know why your mentor is suggesting that as part of the cycle. It makes no sense at all to use a lower annealing temperature than required for a specific product as all you do is encourage non-specific binding and amplification.

Additionally, unless I am reading this wrong, your cycle goes:
denaturation, annealing 55 ,  annealing 63.1, extension

what would happen here is that in the first 30 sec at 55 all of your annealing would be done anyway- so the 63.1 would be pointless...no wonder you are getting non-specific binding.

Ask your mentor for the reason you are using the 55C.

Also, 1 minute for annealing time is WAY too long. Most cycles use 15 to 20 seconds, or even 30 seconds max. But 1 min is overkill.



It is encouraging to see that the amount of primer dimer has drastically reduced, so now you know you can use far less than you were originally.

I know that you obviously have to follow the advice of your mentor, and that you are at her mercy. But to recommend adding more cycles when you are already at 40 is, in my opinion, really bad advice. Yes in theory adding more cycles should allow a faint band to become clearer- but it doesn't address the fact that the PCR itself is obviously very bad if you need this many cycles.

And I am at a loss as to why she won't allow you to do a dilution series with your template. The smearing that you are seeing suggests to me that you may have too much template in your reactions. What is your template, by the way?

(by the way, when you showed your mentor your original photo, what did she say about the bands at the bottom?)

Let me know how you go with this, we'll sort it out

[phage434]

What LeeLee said.

[princeofnam]

Sorry to come back with a reply so late.  I had already scheduled to go home for the summer, sadly right in the middle of my trying to figure out my PCR.  I attempted another PCR with the same settings as above but with a few modifications

1) I dropped down to 40 cycles from 44
2) I ran a PCR Temp Gradient from 58-63 C
3) I took out the 55 C step.
4) Each well had 1.25 ul of forward and reverse primer each.

I'm wondering if the 55 C step was originally in my saved protocol because I started with a temp gradient protocol.  Do you require an initial lower temp for temperature gradient runs?  I'm also unsure about the giant black spot on my PCR.  I any event, lanes 3-11 are my temperature gradient from 58-63.  Lane 5 is my control.  Lanes 3, 7, 9, 11 are control 1, and Lanes 4, 6, 8, and 10 are another control2 .  There seems to be a consistent  band for Control 1, although you can sometimes see it in Control 2.  As always the bottom bands are indicative of rampant primer dimerization which I haven't figured out yet.  There at least seems to be bands from non-specific binding as indicative from my previous post, although I'm not sure either if this one band I'm seeing is non-specific too.  One of the bands I'm looking for is ~611, although other VNTR polymorphisms might be present in 48 bp intervals.  Pretty lost as to what my next run should be.  Perhaps higher annealing temperatures? Lower primer amounts?  Although there didn't seem to be that much of a difference with primer amounts below 1.25 ul in my primer gradient run.

Attached Thumbnails

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Edited by princeofnam, 23 July 2012 - 03:39 PM.

[leelee]

If I am understanding you correctly, you are doing a touchdown- not a temperature gradient.

A gradient means that each reaction tube gets only one annealing temperature...so say you have 8 places that you could put your PCR tube into- the first one would be at 58C, the last at 63C and the machine would do a gradient of temps between those two for the rest.

Eg.

58....59.....60....60.5.....61......61.5......62......62.5......63  (or whatever, it would tell you what the temps are).

What you are doing is a touchdown- where during the run each tube has a few cycles at each temp as the machine runs the cycle from your first temp (58) to last (63). I usually actually run touchdowns the other way (from high to low) but I don't know that that would matter.

In any case, you have removed a great deal of your non-specific binding as the only band present is your desired band (plus primer dimer of course). The smearing suggest to me that you are still running too much template.

Have you quantified your template? How much are you adding to each reaction?

[princeofnam]

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:


Mastermix Preparation:

• 
  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.
  
• 
  Spin Down

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?

[leelee]

So how much template do you add to each reaction then? (amount of DNA, not volume)

What is the concentration of your template stock?

Edited by leelee, 23 July 2012 - 09:07 PM.

[leelee]

princeofnam, on 23 July 2012 - 03:16 PM, 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.

[princeofnam]

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.

[leelee]

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.

Edited by leelee, 23 July 2012 - 09:35 PM.

[princeofnam]

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.
etc.

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.

Edited by princeofnam, 24 July 2012 - 02:42 PM.

[leelee]

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!

[princeofnam]

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!

[princeofnam]

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.