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Digest

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Digest

Postby benedictine » Sun Nov 11, 2007 8:15 pm

hi please may I ask; I did a digest using TaqI; its supposed to have 1,2 or 3 bands according to the polymorphism, but why am I getting sometimes 4 bands and 5. What happened and what should I do to avoid this.
Thank you, from bene
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Postby pluto » Thu Nov 15, 2007 6:52 am

does it have star activity? use less enzyme next time if so.
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Postby benedictine » Thu Nov 15, 2007 2:17 pm

sorry I dont know what is star activity. What is that?
All I know is it is supposed to cut the genes of alleles of a CYP2 that I'm studying. thanks, bene.

also, do you have any idea what to do with acrylamide that has solidified in the beaker (in the refrigerator)? How can we remove it? (I will post this qustion again as a new topic).

thanks again
bene
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Postby blcr11 » Thu Nov 15, 2007 3:00 pm

I usually think of TaqI as a polymerase, so I guess this must be TaqaI? Most likely the extra bands are coming from partial digestion of the restriction sites. TaqaI is sensitive to methylation so if your sites are partially methylated for some reason you may also be getting what looks like partial digestion. I don't think TaqaI has star activity. Some enzymes, when used at high concentration, can recognize additional sites. This extra activity is called "star" activity and the extra recognition sequence(s) is typically indicated with an astersick, hence the "star" designation.
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Postby blcr11 » Thu Nov 15, 2007 5:23 pm

That should've been "asterisk," of course.
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Postby benedictine » Fri Nov 16, 2007 3:53 pm

thanks for writing. It says on the bottle TaqI, not Taqal. Thanks a lot. I use at the most 1ul of enzyme per sample, is that a lot? and from where do we check if our sites are partially methylated? is it from sequence?
thanks again, bene
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Postby canalon » Fri Nov 16, 2007 5:43 pm

I found this enzyme as Taq I with NEB, but it is "Taqa1" on the page:
http://www.neb.ca/detail.lasso?id=R0149&ven=NEB
If you look at the end there is FAQ list, maybe that will be useful, and it says that taq I has some star activity...
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any proof. (Ashley Montague)
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Postby benedictine » Mon Nov 26, 2007 11:01 pm

thanks for the link
from bene
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