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

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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

-lula-

Hi Lula,

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

-pcrman-

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

-lula-

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

-fred_33-

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

-lula-

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

-fred_33-

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

-lula-

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

-fred_33-

hello fred

is BET = ETHIDIUM BROMIDE SOLUTION ?

-lula-

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

Fred

-fred_33-

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