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Old 09-12-2013, 05:05 AM   #1
DonDolowy
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Default MspI digestion - how is it supposed to look?

Hi all,

I am trying to prepare RRBS-seq library, but have some questions regarding the MspI digestion. I find my results a bit odd, since not much is digested and I have not been able to find a representative digestion of gDNA with MspI.

I did a time course (1hr, 2hrs, 6hrs and ON) of 1ug gDNA digested with 20U MspI from NEB, but the shift is not great.

Is the enzyme not working or is this to be expected, since RRBS is only taking 5-10% of the genome into consideration. How come that I also already see a bit of degradation in the lane with undigested gDNA?

Thanks a lot. Appreciate your input.

// Kevin
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Old 09-24-2013, 05:55 AM   #2
C.R.
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here is an example: http://www.zymoresearch.com/epigenet...ylated-dna-set

You probably used a 2% agarose gel to get a nice separation of the 100 bp ladder and the small fragments which you want to excise. Unfortunatley, under these conditions the pattern of the digest looks very similar to uncleaved DNA. Try a lower concentrated gel and you will see a better separation of the bigger fragments.
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Old 10-28-2013, 10:03 AM   #3
DonDolowy
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Hey again, I have now tried several times to digest gDNA with MspI but I never get fragments in the low range (>500bp).
I have tried MspI enzyme from both NEB and Fermentas, which I have tested on commercial human and mouse DNA. Incubation time was 12 hours at 37C.

Since it is just a simple restriction digestion, what can possibly go wrong?

I have attached the bioanalyzer results (high sensitivity DNA chip).
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Old 10-28-2013, 10:13 AM   #4
C.R.
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Hey, the digest just works so good, that I already omit to check it on a gel to safe DNA for some samples. I also used NEB and Fermentas and prepared Libaries with both. If you really want to see the smear after digest: take 500ng to 1 g DNA and incubate at 37C. 2 hours are already enough. Important: put the digested DNA on a 0.7 to 1 percent agarose gel. Do not use the Bioanalyzer CHIP here. If this does not help: can you cut the DNA with other enzymes? Do you really have pure DNA?
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Old 10-28-2013, 10:19 AM   #5
DonDolowy
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Hi C.R, thanks for the quick reply. The DNA I am working on now is completely pure, since it is commercial DNA bought only for optimization to exclude potential problems with impure DNA.
I have also tried to load on a 0.5% gel and I get kind of a smear, but when I continue with the Biooscientific RRBS-seq protocol and do size-selection (300-500bp) using beads, I end up with nothing.
To eliminate rookie mistakes when handling the beads, I asked our sequencing facility to test the protocol, since they are very experienced in library prep for all kind of applications. They as well failed, so it suggests that there really is no small bands.

What is the reason for not using bioanalyzer chips for this purpose? Maybe because the undigested gDNA is taking up such a huge fraction that the chip won't show the small amount of fragmented DNA there might is?

Thanks a lot!
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Old 10-28-2013, 10:29 AM   #6
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Yes, I was also very confused when I looked at my first digest on a 2 percent gel, since I thought that it did not work. Basically what I saw was an accumulation of long fragments which made me think that the digest did not work. I am still doing the traditional gel-excise method, but I think that in principle the bead based method also should work.
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Old 10-29-2013, 08:38 AM   #7
DonDolowy
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I now performed another MspI digestion on commercial available mouse gDNA.

2ug DNA
5uL Cutsmart buffer
1.5uL MspI enzyme
------
Total volume 50uL. 37C, 2hours.

Followed by cleanup using phenol/chloroform/isoamyl alcohol and EtOH precipitation.

I then loaded 1ug on a 0.7% gel, ran 90min @ 100V. The ladder is Fermentas 100bp ladder.

To me there is basically no fragments in the desired range 200-400bp.

I would very much appreciate your inputs!
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Old 10-29-2013, 09:08 AM   #8
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I am not too sure about your image, because the undigested control is missing. In fact I think, that you can see a smear of fragments on your gel picture.

Please have a look at the attached image. MspI was included to the samples on the left side. The right side is the same DNA without any treatment as control. You can clearly see that the enzyme was active and created a smear of fragments ranging from very long fragments to very short ones. Next you see that there is no real enrichment of small fragments visible in this resolution.
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Old 10-29-2013, 11:50 PM   #9
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That gel looks great. Far from what I am getting!
How do you extract your DNA? And are you also using MspI from NEB?
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Old 10-30-2013, 01:50 AM   #10
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Yes it is from NEB. For the gel I used 3g DNA, 5l buffer 4, 2l MspI in a total volume of 50l. 1/3 of the digest was directly used for the gel without phenol-extraction. The gel is 0.7% Agarose in TAE and the ladder is the GeneRuler DNA Ladder Mix (100-10000). I just realized that I am still using an old stock of the enzyme with buffer 4 instead of the new CutSmart version. I doubt that the buffer system change evokes a big difference. But maybe it is worth to ask NEB if this might explain your results.
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Old 10-30-2013, 09:40 AM   #11
DonDolowy
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Finally, it seems like it is working now!

Using a heat block or water bath instead of thermo cycler seems to do the trick. Incredible
No big changes between Fermentas and NEB's MspI enzyme.
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Old 06-24-2014, 12:44 PM   #12
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Very interesting... We are trying the PstI/MspI double digest, and as we are not getting any library that we can detect (due to humongous adapter dimers) we are trying a number of optimizing experiments. Perhaps the waterbath/heat block could be another variable we can use. Thanks for posting
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Old 06-24-2014, 03:42 PM   #13
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Hi rosatoc,

We struggled with RRBS for a couple of months. The input was DNA from 64-cell embryos, so ~200pg. Eventually we ended up using Nugen's ultra-low Ovation kit. We never saw the digestion, but DID see traces after library prep that resembled Nugen's trace @ 200pg. If I remember correctly we only needed 7-8 cycles of amp, in comparison to above 25 in previous attempts. We were observing what looked like adapter concatemers (evenly spaced ladder, unlike MSPI traces), which were verified by sequencing. 99% adapter dimers! Anyhow, we had a couple of small issues with the indexes and sequencing quality, but the library prep was much better. They have a proprietary blunt-end ligation chemistry, but somehow improves efficiency. It's expensive, but they were very generous on a trial kit.
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Old 05-18-2018, 11:16 AM   #14
Castrolsc
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Default Doubts about MspI digestion

Hi all!

I'm having the same situation described before. I'm trying to use the Premium RRBS kit (Diagenode) to analyze bovine DNA samples and it seems the restriction enzyme from the kit is not working well for my DNA (sperm DNA in the picture). I got gDNA from fish testes from another lab which worked for this kit before, and the digestion looks fine.
I'm doing the incubation in water bath (as suggested here before), 37C overnight, and following the kit instructions (100 ng of DNA in 26 uL of water, 1 uL of restriction enzyme and 3 uL of the buffer).
The person who gave me this gDNA recommended to do a clean-up DNA with beads and elute the DNA in water, so I did that for both DNAs, so it should not be a problem related to the sample buffer.
Any suggestions? I tried to look in the literature some profile of bovine DNA digested with MspI but I couldn't find anything, so I don't know what expect.
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Old 05-18-2018, 12:24 PM   #15
JeremyDay
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I hope you mean 100ng of DNA
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Old 05-18-2018, 12:47 PM   #16
Castrolsc
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Yes! 100 ng! Sorry
I corrected the post. And I put all the reaction in the gel, so it should be 100 ng too.
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Old 05-18-2018, 04:05 PM   #17
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@Castrolsc The digestion profile will vary by sample type, but it looks like the digestion for your Sperm DNA worked fine. There is material in the range you need for library generation, and the expected microsatellite bands are visible. Why do you think it isn't working? Have these samples failed when used for library prep?
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Old 05-19-2018, 11:02 AM   #18
Castrolsc
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I continued with the protocol and my Ct values after qPCR were very right (around 19-20), according to the company we should expect something around 10-17, then we started to investigate each step before that and put the digestion product in the gel to see how it looks like. I know that RRBS will only get around 10% of whole genome, but my doubt now is if I could prepare a library with this few amount of bands around 100 to 500bp, because most part of my DNA is >2000bp.
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