Q6: Is MboII affected by methylation?
A6: MboII is blocked by overlapping dam methylation.
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.
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.
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