Restriction enzyme problem

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Restriction enzyme problem

Postby lazyyan » Jun 09 2009 9:20 am

hi admin and chatter,

today, i'm running re to cut my sequence, yes the re was cutting my sequence, my problem is the re was cutting at wrong site. base on http://tools.neb.com/NEBcutter2/index.php, b4 i order the RE, the re should cutting 123, 225 (example). but now base on my gel picture ..the RE not cutting what we aspect in NEB.. i'm using 100 bp marker.

pls give some opinion if u had same problem.. some solutions... thank you
Last edited by lazyyan on Jun 09 2009 1:00 pm, edited 1 time in total.
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Re: Restriction enzyme problem

Postby Lokutus » Jun 09 2009 9:53 am

Which enzyme(s) did you use?

Typical problems are:

methylation sensitivity of the enzyme
wrong buffer used which results in star activity
too much enzyme in too little reaction volume which inreases the amount of glycerol and thus star activity
the wrong template/enzyme used
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Re: Restriction enzyme problem

Postby lazyyan » Jun 09 2009 11:59 am

i'm using Mbo II, my concentration as below, which is total volume 10 ul

sdh20 = 6.5
10x buffer = 1 ul
DNA = 2 ul
RE = 0.5

what do u think?
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Re: Restriction enzyme problem

Postby Lokutus » Jun 09 2009 12:34 pm

Have a look on this page: http://www.neb.com/nebecomm/products/fa ... tR0148.asp


Q6: Is MboII affected by methylation?

A6: MboII is blocked by overlapping dam methylation.



If you try to cut DNA that was isolated from bacteria that encode the dam gene than the DNA is methylated and most probably can't be recognized by MboII. It depends whether the MboII site is surrounded by nucleotides that constitute a DAM methylase recognition site (note, the MboII is a part of the dam recognition site by nature) - if so, then the methylation will overlapp with the MboII recognition site and the DNA can't be cut anymore. This also means that not necessarily all MboII sites are overlapping with methylation which would explain an aberrant restriction pattern.
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Re: Restriction enzyme problem

Postby Onyourcase » Jun 09 2009 5:25 pm

See http://www.fermentas.com/catalog/re/mboii.htm

It says "MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid an atypical DNA band pattern, use the 6X Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis."

Sorted.
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Re: Restriction enzyme problem

Postby lazyyan » Jun 10 2009 5:11 am

more detail, i'm doing RE digestion of PCR products, my extraction from fish
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Re: Restriction enzyme problem

Postby mpagel » Jun 10 2009 1:24 pm

if you're doing PCR, you shouldn't have any two-stranded methylation. The product involving the original parental strands will likely be hemimethylated, but the amount of that product in your overall reaction mixture will be minimal. You can use DpnI post-PCR to get rid double-helixes involving the original template. But that's not where your current problem lies apparently;)

Do you see the 100bp band of your 100bp ladder? Do you get a strong signal from your ladder, or a weak one? Are either of your PCR fragments capped with your target restriction enzyme site, or are the sites internal?

Any chance you can attach a picture of your gel to a post here?
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Re: Restriction enzyme problem

Postby lazyyan » Jun 10 2009 2:02 pm

okey mpagel, i will attach my gel pic as soon as possible in here.

thank you
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Re: Restriction enzyme problem

Postby Iwantcrts » Jun 11 2009 6:52 am

So, your using Fish DNA as a template in PCR, your getting a product of the expected size?
How big is this? How big should it be when cut?
Are you getting a single product/band?
Is it possible that multiple copies of the gene are present within the fish genome? If so mutations may mean that the restriction sites have been removed in one copy but are present in the second, if these genes remain similar in size you`d not separate via electrophoresis, and so it would look like some are cut and other arent.....

Would this explain what your seeing?
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Re: Restriction enzyme problem

Postby lazyyan » Jun 11 2009 9:09 am

i'm using mtdna cytochorome b of fish , more specific, the size product is 334bp (i was already done the sequencing), from the sequencing, i check it on NEB web cutter to get the RE where the RE is more specific on mtdna cytochorome b.

for me this is my first time using NEB RE (mbo 11), and the result was get 2 band/product size. yes, last time i was get 2 band (example 123 and 225) but the RE not cutting at what we aspects or what as showning in NEB WEB cutter.

now i;m planing to do RE concentration , 0.5, 1.0, 1.5, 2.0. mostly i searching they using R.E concentration between 0.5 and 2.0.

i will attach the gel picture in here later.
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Re: Restriction enzyme problem

Postby Onyourcase » Jun 11 2009 3:46 pm

lazyyan wrote:i'm using mtdna cytochorome b of fish , more specific, the size product is 334bp (i was already done the sequencing), from the sequencing, i check it on NEB web cutter to get the RE where the RE is more specific on mtdna cytochorome b.

for me this is my first time using NEB RE (mbo 11), and the result was get 2 band/product size. yes, last time i was get 2 band (example 123 and 225) but the RE not cutting at what we aspects or what as showning in NEB WEB cutter.

now i;m planing to do RE concentration , 0.5, 1.0, 1.5, 2.0. mostly i searching they using R.E concentration between 0.5 and 2.0.

i will attach the gel picture in here later.

We need to nail this baby.
You appear to be saying product size 334bp. Digesting with mboII give 2 fragments 123bp and 225bp?
Sizing small fragments is not that accurate. What makes you think the digest is going wrong? What are you trying to do?
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Re: Restriction enzyme problem

Postby lazyyan » Jun 12 2009 5:49 am

onurcase, here is my detail

my PCR product is 334bp were are showing on the pic below. i have already sequenced.
Image


and base on NEB cutter
Image

where is, Mbo II will be cut at 155bp and 243bp,

here is my first time using RE (mboII), the concentration was

sdh20 = 6.5
10x buffer = 1 ul
DNA = 2 ul
RE = 0.5

from the gel pic the band was below 100bp and below 200 bp ?
Image

and this is my second time using RE, with differences concentration of RE (mboII)

Image

i trying to do FRLP for this mtdna cychorome b

onyourcase: what do u mean "Sizing small fragments is not that accurate." izzit normal?
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Re: Restriction enzyme problem

Postby mpagel » Jun 12 2009 3:26 pm

so, you would expect 3 fragments
155 (start to 155)
73 (155 to 243)
and 91 (243 to end)
assuming complete digestion. (partial digestion could give you a 228bp band and/or a 164bp band)

On the PCR picture, you have the 334 band above the 4th major band. So, what brand of 100bp ladder are you using? Otherwise, is there any chance that you have additional DNA flanking the gene, making a larger product, which would make the first (155bp band) and last (91 bp band) larger? In the top photo, if each band is 100bp and you don't have any bands that "ran off" the gel, I'd estimate the size of your PCR around 440bp. Assigning 50bp to each end would give 205 and 141bp bands in addition to the 73bp band.
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Re: Restriction enzyme problem

Postby Onyourcase » Jun 12 2009 5:27 pm

mpagel wrote:so, you would expect 3 fragments
155 (start to 155)
73 (155 to 243)
and 91 (243 to end)
assuming complete digestion. (partial digestion could give you a 228bp band and/or a 164bp band)

On the PCR picture, you have the 334 band above the 4th major band. So, what brand of 100bp ladder are you using? Otherwise, is there any chance that you have additional DNA flanking the gene, making a larger product, which would make the first (155bp band) and last (91 bp band) larger? In the top photo, if each band is 100bp and you don't have any bands that "ran off" the gel, I'd estimate the size of your PCR around 440bp. Assigning 50bp to each end would give 205 and 141bp bands in addition to the 73bp band.

Yes, so a couple of follow on questions What is the source of the ladder? and Did you remember to include the primers sites in the fragment you searched NEB cutter with? One comment I also have: For a PCR fragment your products are looking really weak on the gel pictures. I can't tell from the pictures but I think if you add much less ladder you will be able to expose for longer and bring up the bands better while still seeing the marker ladder. The gel we need to see has three lanes 1) known marker 2) uncut 3)mboII cut
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Re: Restriction enzyme problem

Postby mpagel » Jun 12 2009 5:39 pm

well...they can be on two different gels as long as the ladder used is the same and the run conditions similar. However, yes, it is best side-by-side, especially as here the ladder looks different gel-to-gel (different pairings of ladder lanes appear to be "merged" or split, and different brightnesses on the final band relative to the other bands)

I had a similar reaction to the strength of the PCR bands. Usually when loading a seemingly similar marker quantity, my PCR bands are usually many times more big and bright than the ladder lane (when I load non-diluted sample).
It's maybe to the point where you should band purify that each "class" of band and sequence it with a forward and reverse primer from the ends. Of course, many of the bands will not amplify at all with one or both primers, due to the fact that one or both sequencing primer sites are missing.
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