Category Archives: Molecular Biology

Ligation, Molecular Biology, Shotgun DNA Mapping

SDM ligation results

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So the good news is that had I not thoroughly messed up the reaction, this would have been a resounding success. The other good news is that this part of the experiment is fairly simple to repeat using the pRL574 anchor. The bad news is the ligation went as I expected it to which is to say sloppily.

Now I have to try to explain to you these results:
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Above is an image of the gel under UV light stained with Ethidium Bromide. I used to use Sybr Safe for visualization (and have in the first part of these experiments), but in the past unzipping was never successful so we think there is a chance that Sybr Safe interacts with DNA differently than EtBr and could hinder unzipping. So in an effort to identically repeat Koch’s grad school work I’m using EtBr because that is what he would have done.

Below is a cropped and grayscale version of the above image:
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We are looking at a lot of lines here. There is a meaning to them all though:

  • The two outer lanes are 1kb ladders in different amounts. More specifically the right most lane has twice as much ladder as the left most lane. We’ll call these lanes 1 (left) and 5 (right).
  • The three middle lanes from left are: ligation with 5′-bio adapter (lane 2) and pRL anchor, internal-bio adapter and pRL anchor (lane 3), and 5′-bio adapter with pALS anchor (lane 4).
  • The ladder reads (from the bottom): 1kb, 1.5kb, 2kb, 3 (brightest band in the ladder), 4, 5, 6, 8, 10kb
  •  Lanes 2 and 3 are identical length wise and the only difference between the molecules is where the biotin molecule is located.
    • The band at the very bottom is the pRL574 PCR product band which is 1.1kb in length.
    • The next two bands above that are pBR322 that has religated into circular DNA.
    • The band above that is digested pBR322 (4.3kb).
    • And the band above that is the unzipping DNA that we want, which is around 5.4kb. I have gel extracted that piece.
    • All bands above this band are chains of pBR322 that ligated together and may or may not have the anchor and adapter ligated to it.
  • Lane 4 is interesting. I would be inclined to say that we got better ligation out of this band because there is less circular DNA bands (compare with lanes 2 and 3). But the product is harder to quantify.
    • The brightest band is the pALS anchor, 4kb in length.
    • Barely above that is the digested pBR DNA, 4.3kb in length. And is identically positioned with the other two lanes.
    • Above that is a very feint band and now I’m inclined to believe that this band represents two pBR322 fragments ligated together and circularized. (Above I say its the product of the reaction that I wanted). I think this because this band lines up exactly with bands in the other two lanes.
    • The next visible band I believe is the product that I expect, 4kb pALS + 4.3kb pBR -> 8.3kb unzipping sequence. It is right there, but again bands in the other lanes could indicate something else.
In orange are the bands that I gel extracted using a razor and incredible wit.

Well tomorrow I’ll gel extract and hopefully we can get some DNA tethers by the end of the week and try for unzipping. Also tomorrow I’ll retry the ligation reaction for pRL-pBR (since I have no more predigested pALS). Tethering is the only real way to identify successful ligation so we’ll find out together!

Ligation, Molecular Biology, Shotgun DNA Mapping

Shotgun DNA Mapping: 3-piece Ligation

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I skipped a whole bunch of steps in the process, but I discovered some prepared DNA from last year that I could use in what is normally the final reaction to make unzippable DNA. The normal process is:

  1. create the anchor via PCR
  2. digest the PCR anchor with BstXI
  3. digest pBR322 with SapI/EarI (EarI creates two fragments which requires a gel extraction, while SapI creates one long fragment)
  4. 3-piece ligation with anchor, unzipping fragment, adapter

The DNA I found I named BpALS, BpRL, and SpBR. The nomenclature is a little strange but the B stands for BstXI, which means those DNA pieces (BpALS and BpRL) are digested with BstXI. The S stands for SapI which means SpBR has been digested with SapI.

Today I set up a ligation reaction using this old DNA (since DNA is really stable and stays usable for long periods of time) and using the new adapters I purchased. Check out the reaction below:

This reaction is different than a normal ligation reaction. In this reaction I start with a small concentration of adapter compared to the concentrations of anchor and unzipping DNA and slowly add more adapter. The reason for this is because I want to ensure that the ligation creates one continuous piece. If I add equal concentrations of all three pieces, then I could end up with a lot of adapter ligated to both the anchor and the unzipping DNA. Since the adapter is designed to not ligate to itself this would create two fragments that cannot ligate to each other.

NOTE: There is a really good chance I sabotaged this experiment. Digesting pBR322 with SapI produces a singular and linear DNA product because it cuts in exactly one location. The problem is that when ligating, this may self ligate. I normally do a ligation with SpBR and the adapter with the adapter in tremendous excess to prevent this. Then I purify that reaction and do another ligation with this new product (that I call ApBR for adapter-pBR322) and the anchor piece. Today I forgot to do this first ligation reaction and went straight for the three piece method which was designed for the EarI-digested unzipping fragment otherwise known to me as EpBR. Hopefully the gel analysis reveals some good news.

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 6: results

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Well both reactions from yesterday failed. I’ll have to do a Mg++ titration to see if I can optimize the reaction at the original temperatures.

In the meantime I do have enough to move on and go to the digestion and ligation reactions. Moving on…

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 6: setup

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I’m making slight modifications to the last reaction before I move on:

  • I’m changing the ThermoCycler program temps just a little bit based on some observations I made with the machine yesterday. I noticed that running the machine on manual isn’t the same as running a program, during a program the block actually will go over (or under depending on the situation) temp while the machine measures the temp of a tube with oil in it. My measurements from yesterday did not experience this in manual mode.
  • I’m also trying OneTaq in OpenPCR with some slightly modified temps, hoping that helps the reaction a little bit.

Here are my reactions:

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 5: results – SUCCESSFUL

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Here is the setup from yesterday.

I ran a 0.8% gel prestained with Sybr Safe and viewed with the invitrogen illuminator for this stain.

And below is the image of the gel taken with my crappy-ass camera phone (Droid Bionic).

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Yay! The reaction finally worked! Well mostly. It worked in lanes 6-10, which correspond to one of the reactions from the OpenPCR and 4 of the reactions from the ThermoCycler. I will assume the 5th reaction worked as well. That reaction is not visualized because there are only 10 lanes in the gel and there are 10 reactions plus the DNA ladder so one would need to be left out.

I did a reaction cleanup with Novagen PCR Cleanup. I couldn’t find my Qiaquick PCR cleanup kit and this was all I thought I had. After cleanup, I found my Qiaquick kit and will use that next time.

The nanodrop says there are 35.3ng/ul of PCR product which correlates to ~13nM. That isn’t great but it’s a start. Moving on… FINALLY!!!

Molecular Biology, PCR, Shotgun DNA Mapping

Thermo PCR Sprint (“ThermoCycler”) Programmed T vs Recorded T

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This experiment is a follow up to yesterday’s experimental results. Instead of trying to program the ThermoCycler by guessing, I decided to record the temperatures of various different T settings. Basically I would pick a T and try to get the recorded value near the T’s needed for the pALS PCR protocol.

I put the machine in manual mode so I can change the T when I needed to and I would take data points at 30 seconds for melting and annealing temperatures and at 30s and 5 minutes for extending T. This is how long I would be doing that step during the PCR reaction so it seemed to make sense. Below is a chart of the Set T and the recorded T at each interval.

Below that is the data of the recorded T.

The recorded temperatures of the PCR Sprint thermal cycler.

Figshare Data:

PCR Sprint Programmed T vs Recorded T. Anthony Salvagno. figshare.
Retrieved 18:51, Aug 23, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.94414

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR Try 5

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SO I’ve done the temp experiments and decided to do a quick temp reading of the Thermo Sprint PCR machine this morning. That data will be up shortly. The temperatures for the PCR reactions below are based on those readings, while the T for the OpenPCR reaction (also below) are based on the readings from the experiments the past few days.

Anyways now I have two machines that are better prepared to perform this PCR reaction. Both machines are set for actual temps of ~92C, 50C, 72C and hopefully I get something that I can work with today. Reaction setup is here:

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.