small sized restricted bands couldnt be visualized - restriction enzymes problems (Mar/04/2005 )

Lula,
I run the MTHFR A1298C on a 3% agarose gel for 30 min @ 100 V. Fred is right Agarose is quick, cheap, and easy. And you can peak at your run before the 30 min is up with no problem.
When you don't cut w/ MboII do you get a band at 163 bp? Are you having trouble geting bands on heterozygous muts, homozygous muts, and normals after cutting w/ MboII?
Jim

hello

yes Jem im getting the original PCR product seen on 2% agarose
its about 168 bp ....but after i add Mbo II i dont see anything
not on agarose 3% and not on polyacrylamide 20% ....the gel appears clean from any thing
im using for the restriction :

25ul Pcr product
1ul enzyme
3ul buffer
1ul water

whats wrong ??
NB Fred :thanks for telling me about the BET

Lula,
Could you have over cutting due to relaxed specificity of the enzyme? Excess enzyme or excess glycerol (buffer) would cause this. I don't use buffer, I use 0.5 ul enzyme to 25 ul pcr product. I use New England biolabs; the concentration is 5,000 U/ml (I use 2.5U/25ul).
There could be another enzyme in the tube w/ the MboII, have you gotten the assay to work on any control rxns?
Also nuclease contamination could cause you to have no DNA after cutting.
Hope this helps,
Jim

thats new to me to use the enzyme without a buffer ?
thats nice i well try it for sure
u mean u put the enzyme +pcr but no buffer and no water ??
for how long do u incubate the product after adding the restriction enzyme?
i incubate mine for 16 hous then freeze

hi
i think that 16hours of digestion is a long time. Usually i make 2hours of digestion. Neb didn't report star activity on Mbo II but maybe you get it in your reaction conditions...

I recommend you to do shorter times of digestion and see.
But i never heard about digesting whithout buffer and you can incubate for a short time let say 15' and add digesion enzyme

good luck
Fred

After I run the pcr rxn, I add 0.5 ml of the restriction enzyme (5000U/ml) to each rxn tube. So the only buffer comes from whatever NEB puts in the tube with the RE. I don't know how, or why, this technique was initiated; my predicesor was not around when I came on board. It worked, so I kept it.
I digest for around two hours, but I have digested for up to 16. If I'm too busy to run the gel I freeze but usually I run them right away.

HELLO


i well try to put th restriction enzyme with different concentrations starting from ,5 ul till 1ul and incubate for only 2 hours in 37 C incubator after adding the enzyme

by the way whats ur annealing temp Jim?? and do u have photos of what bands to see in the hetero and homozygotes after restriction ?
thanks Fred and Jim

Lula,
My rxn temps are:
95 C-2min
(94C-30";65C-30";72C-30")X35
72C-4min
I have photos; how do we go about sending pictures within this forum?
Jim

hi Jim


thanks for replying
i m new to this forum so i dont know how to send photos at it ...but if it would help u can send the photos as attatchments on my email
dr_genome1@hotmail.com
if its not so much trouble
thanks again

No problem, I'll send them from my home computer; I don't have a scanner here and the're polaroids.
Hope all is going well,
Jim