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small sized restricted bands couldnt be visualized


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22 replies to this topic

#1 lula

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Posted 04 March 2005 - 12:52 AM

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

#2 pcrman

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Posted 04 March 2005 - 06:43 AM

Hi Lula,

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

#3 lula

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Posted 04 March 2005 - 08:44 AM

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

Edited by lula, 04 March 2005 - 09:12 AM.


#4 fred_33

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Posted 07 March 2005 - 01:23 AM

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

#5 lula

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Posted 07 March 2005 - 07:45 AM

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

#6 fred_33

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Posted 07 March 2005 - 08:14 AM

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

#7 lula

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Posted 07 March 2005 - 01:20 PM

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

#8 fred_33

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Posted 08 March 2005 - 12:35 AM

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

#9 lula

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Posted 08 March 2005 - 07:49 AM

hello fred

is BET = ETHIDIUM BROMIDE SOLUTION ?

#10 fred_33

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Posted 08 March 2005 - 07:57 AM

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

Fred

#11 pcrpetie

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Posted 09 March 2005 - 05:52 AM

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

#12 lula

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Posted 09 March 2005 - 10:54 AM

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

Edited by lula, 09 March 2005 - 10:55 AM.


#13 pcrpetie

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Posted 09 March 2005 - 12:00 PM

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

#14 lula

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Posted 09 March 2005 - 01:48 PM

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

#15 fred_33

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Posted 10 March 2005 - 01:10 AM

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

Edited by fred_33, 10 March 2005 - 01:12 AM.





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