Greetings, everyone!
I've been testing my restriction enzymes, and have candidates for a rare cutter and a common cutter. The rare cutter is BamHI, and my frequent/common cutter is PstI.
I tested each enzyme separately and together (B+P = BamHI + PstI). Each digestion was allowed to proceed for ~ 6 hours, and reaction conditions were set according to the manufacturer's recommendations.
I ran these on gels next to undigested genomic DNA; I've attached the results below.
My concern is that there is still a band present in the BamHI lane, though it appears in both cases that it is slightly smaller than the undigested genomic DNA (the ladder is Hyperladder I; sorry for the over-exposure).
Is this especially problematic, or is this something that is commonly seen when performing these types of digestions with "rare" cutters?
I will note that I am aiming for a highly reduced-representation library; my organism's genome is ~ 8 Gb in size, and I'm trying to maximize coverage - so an enzyme that doesn't cut very often is desirable. But this has me wondering whether this one is cutting too infrequently (if at all)?
Many thanks,
Sean
I've been testing my restriction enzymes, and have candidates for a rare cutter and a common cutter. The rare cutter is BamHI, and my frequent/common cutter is PstI.
I tested each enzyme separately and together (B+P = BamHI + PstI). Each digestion was allowed to proceed for ~ 6 hours, and reaction conditions were set according to the manufacturer's recommendations.
I ran these on gels next to undigested genomic DNA; I've attached the results below.
My concern is that there is still a band present in the BamHI lane, though it appears in both cases that it is slightly smaller than the undigested genomic DNA (the ladder is Hyperladder I; sorry for the over-exposure).
Is this especially problematic, or is this something that is commonly seen when performing these types of digestions with "rare" cutters?
I will note that I am aiming for a highly reduced-representation library; my organism's genome is ~ 8 Gb in size, and I'm trying to maximize coverage - so an enzyme that doesn't cut very often is desirable. But this has me wondering whether this one is cutting too infrequently (if at all)?
Many thanks,
Sean
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