Measuring Pyrimidine Dimers in Planaria

[mserrano]

Hi!

Thank you so much for the link! I will look into it, and see if I have the available resources.

One question I have about the T4 endonuclease V is, will it cleave the DNA only at the pyrimidine dimers or will it cleave at other places as well? I plan to quantify the DNA by seeing whether or not the DNA will be cut into different lengthed segments at the pyrimidine dimers, and compare it to normal (control) DNA in order to see if the UV truly created damage in the planaria's DNA. But will there be other problems that I will run into while using the T4 endo V that will interfere with the quantification of the DNA that I would like to do (i.e. the T4 endo V will cut at points other than the pyrimidine dimers)?

Thank you so much,

Madison

[SciB]

Hi Madison,

I can answer your question simply--no, T4 endoV only cleaves the DNA at pyrimidine (T-T) dimers. So, there will be no other strand breaks.

Here's a classic paper on the enzyme that states specifically that it only cuts at dimers and that it works most effectively on normal double-stranded DNA: http://www.pnas.org/content/67/4/1839.short

As it says in the paper, the number of cuts in the DNA is equal to the number of T-T dimers so you really need to do some reading about UV doses and how strong they have to be to create a certain number of dimers. What are you using as a UV source? Do you have a way of measuring the UV energy that you will be delivering? This is something that you really should determine but it requires a special meter that you may not have. There is also a way to measure UV dose with test strips. Here's one example but there are others: http://www.uvprocess.com/product.asp?code=INTS+LBL+B

Remember that UV light can damage your skin and especially your eyes, so whatever source you use wear safety glasses and keep your hands away from the light when it is on. You could set up the lamp inside a box so that the light including reflections is contained and blocked.

Hope this helps,

Sybee

[mserrano]

Hello,

I am currently in the process of ordering my supplies for my experiment, and I was wondering where/who is the best supplier to order the T4-Endonuclease V? I currently have this link, and i am considering ordering it from here.

https://www.neb.com/products/m0308-t4-p ... nuclease-v

Thank you!

[SciB]

Hi Madison,

Yes, New England Biolabs is one of my favorite supplies. Their quality is excellent and the prices are reasonable. One thing that you could try is to call them and tell them you are a student doing a science project and would like to know if they have a student discount or if they will sell you a vial of endoV that is near or past its expiry date if they have any.

It certainly can't hurt to ask for a student discount. Your school can FAX them a verification if they need it.

I don't know if NEB keeps any of their products that are close to the expiration date, but that is something else you could ask about if they don't give a student discount. We often use expensive enzymes past their expiration date with no problem.

Let us know how you make out. Your feedback on a project is very important as it helps us to be able to give others better information.

Good luck!

Sybee

[mserrano]

Hello!

That sounds great! I'll contact them and inquire about a student discount, and check in again here.

I have one more question, how should I add the T4 endo V to the extracted DNA? I will be using a Qiagen DNeasy Blood/ Tissue DNA extraction kit, which follows these directions:

https://www.qiagen.com/us/resources/dow ... be&lang=en

At which point in the procedure will I add the T4 endo V, and what amount?

And after which adding the T4 endo V, can I put the sample through electrophoresis right away?

Thank you so much!

[SciB]

Hi,

Have you read some scientific papers on pyrimidine dimers and how researchers detect them? That is how you should answer your questions. I don't know the answers to your questions right off so what I would have to do is search for the procedures online and read them until I found the answers, which is exactly what you can do. Also the companies that sell enzymes like endoV have data sheets on their websites that give you the methods for using the enzyme. And they have tech support teams to answer your specific questions.

Try coming up with a procedure yourself and then post it and I will check it. You will learn a lot more by doing things yourself. Reading and learning new methods are all part of scientific research.

Sybee

[mserrano]

Hello,

I'm well into my experiment now, and I've gotten quite mixed results. My biggest problem is extracting DNA from the planaria. I've been using the Qiagen DNeasy Tissue extraction kit, and I just haven't been getting any results. At first I tested it on one worm, then when that did not work I tried 5 worms, and even went up to 20 worms in one sample, but none of them resulted with even the faintest DNA band after running through gel electrophoresis (1% agarose gel). My teacher suggested I try amplifying a specific fragment of the planarian DNA through PCR, but I am not sure which primers to use for this, so if you have any suggestions, this would help greatly. Also, is there any other tested procedures that are known to extract a significant amount of planarian DNA?

Another problem I'm experiencing is regarding the T4 endonuclease V digest. I decided to test my procedure on plasmid DNA (pGLO), in which I exposed 10 μL of the plasmid to 280-320 nm of UVB for 2 minutes, and then I added it to the endonuclease digest solution (10μL endonuclease, 10μL reaction buffer, 5μL BSA, and 20μL water), and finally I let it incubate at 36 degrees celsius for 16 hours. I repeated this procedure on a sample of plasmid that has not been exposed to any UV for the control. When I ran the samples on the gels, the DNA was streaky on both the control and the exposed samples, and neither my teacher or I know exactly why this has happened. Photos of the gel scans can be found at the link below:

https://docs.google.com/document/d/132e ... N1fE0g/pub

Any suggestions or opinions about my procedures or any explanations to the problems I am facing would be greatly appreciated.

[SciB]

Hi,

Sorry you are having problems, but that's actually pretty normal in research. Let's see if we can figure out why you are not seeing any planarian DNA.

Thinking about the process I can see two areas where there may be a problem:

1. DNA extraction
The DNeasy kit is very reliable but the instructions have to be followed to the letter. You also have to have a centrifuge with the correct speed. Since I can't watch you performing the extraction there's no way to guess where there might have been an error. You can call Qiagen's tech support and ask them for help.

2. Agarose gel electrophoresis and detection
There are several things that can go wrong here. What buffer did you use to make the 1% gel? What electrophoresis apparatus are you using? Did you include a tracking dye such as bromophenol blue? Did the dye run properly? What voltage did you run at and for how long? Did you also run DNA size markers?

What volume of your DNA did you load? Are you sure it went into the wells ok? How are you detecting DNA on the gel? Did you run some known DNA, like your plasmid, to make sure the detection system was working properly? Using a tracking dye and DNA control are necessary for every run. Do you have access to a UV spectrophotometer? I always check the absorbance at 260 nm and 280 nm of my DNA before I run it to make sure that it has a 260:280 ratio of 1.7 to 1.9 indicating a good extraction. You can't do this without a UV spec, but it is the one step that guarantees that you have good DNA and all researchers would check their DNA before using it. If you could possibly find someone with a UV spec that you could use it would be very helpful.


With regard to your test of the T4 endoV, what is the concentration of the plasmid DNA? You said 10 uL but that does not tell me how much DNA was in that 10 uL. Where did you get the plasmid? Can you be sure that the DNA is not degraded already? If it looks 'streaky' on the gel it may be no good. How large is the plasmid? Did you run DNA size markers?

I've asked you a lot of questions, so please answer them and I will try to pinpoint what went wrong so you can get on with your project.

Sybee

[mserrano]

Hello!

Thank you so much for your reply! I will try to answer your questions.

To make the 1% agarose gel, I used 100 mL of a 1X TAE buffer, which I added to the 1g of agarose (QS to 100 mL in bottle). As for my electrophoresis apparatus, I am using the Bio-Rad Sub-Cell GT Agarose Gel Electrophoresis System. In my first trial, with only one worm I ran the gel for 25 minutes at 100 watts, and in my trials with the 5 worms and 20 worms I ran the gel for 45 minutes at 100 watts in order to allow it to travel a little further down the gel. In terms of the tracking dye, I used the Bio-Rad UV View 6x Loading Dye. I also used the Bio-Rad EZ Load 1 KO molecular ruler 80 micrograms/mL.

As for the volume of DNA that I used, I just measured 10 microliters of the Bio-Rad pGLO Plasmid Lyophilized DNA. I first ran the pure plasmid dna on the gel (no digestion) with the UV tracking dye through the gel just to make sure my apparatus was working properly, and it passed through and showed up on the gel fine (clear bands). But once I added the T4 endo V digestion solution, the bands turned out in the smudged way that I described.

Hopefully this answers your questions, and I look forward to your further advice.

Thank you!

[SciB]

So, when you run the plasmid by itself it is fine but when you digest it with the endoV it gives a smear. I would call the company that you bought the endoV from and talk to tech support. The T4 enzyme is not supposed to cut DNA that has no T-T dimers. There may be something wrong with that batch of endoV in which case they should send you a new vial free.

I believe you said you were going to buy the endoV from New England Biolabs so I checked their tech info on the product and found this warning in their Notes (https://www.neb.com/products/m0308-t4-p ... nuclease-v):

Notes
For best results incubation time should be 30 minutes or less at 37C.

You said you did the digest for 16 hrs. I checked the product instruction sheet and I see where you got that--under the Quality Control section. This was where they tested the endoV for non-specific activity by doing a 16 hr digest of lambda DNA. They report no degradation but that is under their conditions. I suspect that your plasmid solution may have a little nuclease activity which does not show up when you just run it on the gel but if you incubate it for 16 hrs with the reaction buffer which provides the nuclease with factors it needs to work, you get degraded DNA. You should test this by incubating 10 uL of plasmid with reaction buffer and BSA but no endoV for 16 hrs. If it is degraded then there are nucleases present. Try doing the digest for only 30 mins and hopefully it will work.

You still haven't told me if you measured the concentration of your planaria DNA and what it was. You first need to know if your DNA preparation was good and to do that you need a UV spectrometer to measure the absorbance at 260 and 280 nanometers. Then you get the ratio 260/280 and it should be around 1.9 if your DNA prep is good. Assuming you have a good ratio, now you can determine the DNA concentration in your isolate. Here's a reference on how to do that: https://www.promega.com/resources/pubhu ... na-sample/

Good luck!

Sybee