Category Archives: PCR

Molecular Biology, PCR, Shotgun DNA Mapping

PCR machine testing: results

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Via figshare:

OpenPCR and Thermo PCR Sprint Thermal Cyclers testing. Anthony Salvagno. figshare.
Retrieved 22:53, Aug 22, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.94408

Well, well, well. It looks like OpenPCR works much better than my ThermoCycler (which I just learned today is actually called PCR Sprint). The temperatures match pretty well between what the program is set for and what the recorded values are. I’ve discovered in the past that OpenPCR has a problem getting above 90C (see bottom of post), but I don’t think that is an issue here. If OpenPCR isn’t producing product then I may not have chosen the right annealing/extending temps or the problem may be elsewhere (unlikely).

ThermoCycler on the other hand is a piece of garbage. I’ve always hated this thing and now I have proof (again, I’ve done an experiment like this in the past, but it wasn’t as bad as these results show). Looking at the file “thermo-oil-3-cycles.png” you can see that ThermoCycler never gets below ~62C which is sad because it is set to anneal at 52C. It also extends at ~74C when it should be near 69C.

Tomorrow I’ll be taking some T measurements to try and get the program to be closer to the temps I want to run the pALS protocol at. OpenPCR will just need some slight modifications, but ThermoCycler needs a lot of help. Sigh…

Molecular Biology, PCR, Shotgun DNA Mapping

PCR machine testing: Day 2

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I can only run two PCR reactions in a day, and originally I planned for four, but now I’m going to be doing 5. Why?

  • Well first I ran the OpenPCR pALS program with mineral oil in the tube and found out the connections were faulty so the data collection was erroneous (I would get values like -78C).
  • Then I ran the ThermoCycler pBSTXI program (pBSTXI is the original name for my pALS plasmid) with water in the reaction tube and the water evaporated!
  • Next I ran the OpenPCR pALS program again, this time with water in the reaction tube, and that water evaporated as well.
  • Now I’m running the ThermoCycler pBSTXI program with oil in the reaction tube to compare the results.
  • Tomorrow manana (that’s morning for the non-Spanish speaking) I’ll be running the OpenPCR pALS program one last time with oil in the tube again to get better data than the first run.

I’ll be publishing all the data sets at once even the bad data, because it’s open science and I can’t be open if I’m not 100% open. It’s all or nothing. Or at least that’s what my brain says the rules are.

Here is a sneak peak at the data (full data will be posted to figshare tomorrow):

Molecular Biology, PCR, Shotgun DNA Mapping

Testing the pcr machines

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Today is going to be dedicated to getting temperature readings from the PCR machines (both OpenPCR and the ThermoCycler). Both machines have their own temperature output, but in the past I’ve discovered that they don’t really reflect the temp inside the PCR reaction tube (especially with regards to the ThermoCycler). The last time I did this with the ThermoCycler, I got some interesting results which led to my current program settings.

Today I’m going to repeat that experiment and replicate it with OpenPCR. Here is my setup:

  • You will need:
    1. a PCR machine
    2. reaction tubes
    3. a thermocouple – These are pretty easy to find
    4. a temperature reader – I’ve used digital multimeters in the past, but for this experiment I’m using the TC-48-20 OEM because it comes with software that takes frequent measurements and allows me to export the data.
  1. Take a PCR reaction tube and drill a small hole in it
  2. Put either water or mineral oil in the tube (in the amount that you normally use for PCR reactions), I’m doing 50ul for this experiment.
  3. Put the thermocouple in the tube through the drilled hole and place the tube in the block on the PCR machine.
  4. Connect the thermocouple to your temperature reader (in my case I need to connect the temp probe to the TC-48-20 and then connect that to my computer)
  5. Run the PCR program and collect data.

Check out the images below:

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From right: a small drill bit, a PCR reaction tube, and a 15k thermocouple
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Once you drill the hole, add some water (or mineral oil) to the reaction tube, and then put the thermocouple in through the drilled hole.
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The temperature controller with only the temp probe and computer connected
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The connection between the temp probe and the temp controller.
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The entire setup: OpenPCR, temp controller, computer with software.
Molecular Biology, PCR, Shotgun DNA Mapping

Notes about PCR

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I took these notes a long time ago about PCR and how to optimize for it and what each component needs. The notes are taken from the book Molecular Cloning, which is just full of amazing detail and is a must for any lab. Thought I would document this for myself (or anyone else) for that matter.

I realized that last week was a pretty wasted week. I hadn’t done PCR in a year and even though equipment shouldn’t just cease function, I should have started checking the equipment to make sure everything was working properly. I also have been just picking possible errors and adjusting instead of doing a systematic approach to correcting my PCR issues. Well my blind aiming is over and now I’m going to troubleshoot the PCR reactions the right way.

Molecular Biology, PCR, Shotgun DNA Mapping

PALS PCR 4 results

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image

  • 0.8% gel: 50mL of 1x TAE, 0.4g agarose, prestained with Sybr Safe
  • out of 10 reactions only 1 reaction worked… hmmm. And there is a chance that the product isn’t even what I want. It appears that the band is not 4kb, but the gel didn’t run evenly so there is a chance it is 4kb but doesn’t appear that way because of how the current pulled the DNA.
  • It looks like a machine test is in order. The Thermo cycler said the program completed today (which it hadn’t in the past few trials), but also said the program completed in 2.5 hours (which it shouldn’t because it is a 4.5 hour program). And the OpenPCR won’t get below 16C for the final hold even when I set it at 15C, which it should not have a problem holding at (I’ve seen it consistently get to 12C).
  • I will also order new dNTPs even though I think that may not be the problem.
  • I also learned yesterday that the freezer wasn’t closing completely and everything may have thawed potentially ruining the enzyme.
  • Finally I should try to mix the master mix better if that is an issue at all.

Lots to do in so little time… Gotta get this working by this weekend.

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 4: The switcheroo

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I’m doing almost the exact same experiment from yesterday. Today I’m just putting the 1Taq reaction in the Thermo thermal cycler (Thermo cycler anyone??) and the Taq reaction in OpenPCR. Perhaps the temps aren’t quite the same and work better in the other machine. This made sense to me before I set up the experiment… Anyways here are the protocols:

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 3 results

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Blah! I grow disdain for failed PCR reactions. I have no idea what the issue is and I hate troubleshooting PCR. Oh well. I’ll stop venting and get right back to work.

So the PCR was a failure again. And now I have to figure out how to adjust it. Since I’m not getting any product here are my potential fixes:

  • adjust the annealing temperature
  • try different amounts of MgCl2
  • try new dNTPs

In order to properly adjust the annealing temp I would need to make sure the machines are functioning properly, which I’m figuring they aren’t. The reason I think this is because when I check the thermal cycler (not OpenPCR) it says Program end in 0:00 and usually says Hold 4C. But the reason I think the thermal cyclers are not the issue is because neither of them produce product, their reactions are identical, and their temps are very similar.

In order to troubleshoot the machines I’ll have to run a temp experiment where I track the temperatures over time. I have equipment for this, but I can’t find all the components. I’ll have to talk to Pranav about the missing parts.

Trying different amounts of MgCl2 is easy. I would just do amounts ranging from 1 to 5mM of MgCl2 all on the same program.

In order to try new dNTPs I would just need to buy new dNTPs. This can’t happen until Monday. Neither can the thermal cycler experiment. Tomorrow I can try a MgCl2 titration experiment. Right now I’ve got a simple check in mind.

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR adjustments

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So I just noticed that my reaction program was set with an extension temperature of 65C and it should have been 69C according to my notes. Adjustment made. I will then be setting up the reaction as shown below. It should be noted I’m doing an OpenPCR run with OneTaq (from NEB) and a regular PCR reaction (with Taq in the regular thermal cycler). Here are my protocols:

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 2 result

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Gel setup:

  1. 50ml of 1xTAE buffer with 5ul of Sybr Safe (10000x in DMSO) and 0.4g of high quality agarose
  2. microwaved for 2 min
  3. poured into electrophoresis apparatus with 10 well comb (from Owl)
  4. running at 150V for ~40 min

Results:

So the PCR reaction didn’t work again. Hmmm. My first measure of adjustment is usually to adjust the annealing temperature so I’ll start there. Troubleshooting PCR is so annoying, but I want to get some product before next week so work at it I will.

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR OpenPCR vs Thermal Cycler 2

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Before I set up my reaction, I need to dilute my oligos from IDT from 100uM to 10uM, and dilute my pALS plasmid from 160ng/ul to 1.5ng/ul or basically a 1:100 dilution.

You may have noticed that this is the same thing I did last week. The difference is I’m using new oligos, DNA, Taq, etc. Hopefully the reaction runs better. Here is my reaction: