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

hello everyone

im new to this forum but i thought may b someone here could help.im having trouble in detecting the restricted fragments im working on
im working on the MTHFR 1298 A- C polymorphism using MboII restriction enzyme .seems i have good PCR product at first but after restriction enzyme application i dont get any results at the 20% polyacrylamide gel im using .
if any one has useful information could u please send it to my email dr_genome1@hotmail.com

thanks all
lula

Hi Lula,

What is the original size of your PCR product? How many new fragments after digestion and what are their size?

hi



six fragments of 84, 56, 31, 30, 28, and 18 bp, should be produced fter the digestion
im supposed to see th 84 bp and the 56 bp ...but i dont get any product after using the restriction enzyme

hi
maybe you can try a 4.0% BET agarose gel ? it is great for separating these fragment sizes (but a little bit difficult to dissolve...)
Fred

hi fred
i can use the 4% agarose but may i ask what the word BET stands for ??
thanks

hi
BET is for visualize your DNA fragment. It inserts itself between bp and is fluorescent under uv light. You add 1µl of 10mg/ml BET solution to 100ml Agarose solution. BET supports further microwave-oven utilisations.
Fred

hi
do u think that there is any chance that i can visualize these bands on 20% polyacrylamide gel ?????
i did try them once on polyacrylamide with eithidium bromide but no luck
i always get pcr products (checked on 2% agarose)but after using the restriction enzyme i get empty lanes

hi
certainly, if you have enough quantity if your digest products, they should be vizualized on 20% acrylamide gel. But i recommend a denaturing 7Murea/20% PAcryl gel.

i think that agarose gel is more easy to prepare. It's you to see.

Fred

hello fred

is BET = ETHIDIUM BROMIDE SOLUTION ?

hi
yes BET = ethidium bromide solution. I wrote BET 'cause i use this abreviation in french. Sorry. Next time i will explicit better.

Fred