Category Archives: Molecular Biology

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:

Molecular Biology, Shotgun DNA Mapping

Annealing the adapter oligos

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I purchased some oligos and now I need to create the adapter duplex from those oligos. This requires an annealing reaction which is super simple to do. Basically you just mix your oligos together, heat them to 95C and then slowly cool the mixture. Nature takes care of all the leg work. I’ve done this reaction 3 different ways:

  1. Heat a cup of water to boiling in a microwave, then remove the water, place your annealing mix in the water (make sure the top is floating), and allow the water to cool on a lab top. Basically allow the water to cool to room temperature (RT).
  2. Put your mix into a heating block and heat that to 95C (or close to boiling). Once the mix has been heated, remove the block from the heating unit and place on a lab top to cool to RT.
  3. Put your annealing mix in a PCR machine which can control the temperature very specifically. Create a program that will: (1) heat the mix to 95C for about 5 minutes, (2) slowly lower the temperature to about RT or 4C or whatever cool temp you want, (3) hold at that low temp until you are ready to remove it and move on.

For today’s reaction I will be using option 3. My protocol for the experiment is below. Unfortunately there is no easy way to verify the annealing reaction is successful. Well that’s not 100% true. You can run an SDS-PAGE gel which has the ability to resolve very short DNA sequences, but I don’t really have the equipment for that right now. I do have a high resolution gel that I’ve been wanting to try. Hmmmm…

And here is a link to what my annealing buffer is: Annealing Buffer Recipe.

Molecular Biology, sequences, Shotgun DNA Mapping

Resuspending Oligos from @idtdna (with resuspension calculator)

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I created myself a resuspension calculator that calculates the actual measured concentration from what it should be. IDT has a resuspension calculator that seems to just take the nmole amount and divide it by 1000 to give you the amount to add in ml. My calculator does the same thing, and allows me to input nanodrop values to get a nanodrop measured calculation. Here are the calculations for these oligos:

Also if it isn’t apparent from the spreadsheet, my resuspension buffer is just 0.1x TE buffer. All calculations are in the spreadsheet which is publicly available to make copies of as you see fit.

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR Results OpenPCR vs Thermal Cycler

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Yesterday I setup a PCR reaction using both our labs thermal cycler from Thermo and our OpenPCR thermal cycler. I want to make pALS PCR fragments (4kb in length). And here are the results of that experiment (with setup):

Gel Setup:

  1. 50ml 1x TAE buffer mixed with 0.4g High Resolution DNA agarose
  2. heated in microwave for 2 min
  3. added ethidium bromide (EtBr) to final concentration of 1ug/ml
  4. gel cooled for 40 min in fridge
  5. Meanwhile, 5ul of 9 of 11 PCR reactions put in PCR tubes with 1ul of 6x loading dye added (for 6ul total per tube)
    1. the tubes selected were 4 tubes from OpenPCR (numbered 1-5) and 5 (of 6) tubes from Thermo thermal cycler (numbered 6-11), so tubes 1-4 and 6-10 were selected for gel analysis. This is because there are only 10 wells and 1 well is needed for DNA ladder.
  6. Once gel cooled, 250ml of 1x TAE added to electrophoresis device (what’s the name of this thing?), filled to cover gel
  7. 6ul of each of the 9 prepared tubes were placed in the wells along with 6ul of pre-prepared 1kb DNA ladder.
  8. connected to power supply and run at 150V for 45min

Results:

image
Gel results. Lanes 2 and 4 have feint bands where the expected PCR result should be.
image
Enhanced gel results.

Based on the images above, the PCR reaction was a failure. Lanes 2 and 4 have very feint bands where the 4kb product should be, indicating the reaction succeeded but not to the degree required. This could be for any number of reasons, but most likely due to inactive enzyme, old reaction buffer, degraded dNTP’s, etc. Basically I’ll be doing this reaction again next week when all my new supplies come in.

Notes about the image acquisition:

Since I ran the gel with the EtBr added to the molten gel, I did not need to run the gel and then add EtBr. I also did not destain the gel after completion of electrophoresis. The images above were captured with my phone camera. To see gel results typically I use SybrSafe from Invitrogen with their special illuminator, but since I used ethidium today I had to use the hand held UV lamp we have. This means worse quality photo, but oh well.