high percentage agarose

Hi there,
I am a PhD student working on Pneumocystis biodiversity. I cam across
your message posted on google groups, regarding high percentage
agarose gels and TBE concentration. One of the objective of my thesis
is to do typing by counting number of 10 base pair repeats in my PCR
products. The number of repeats can vary from 2 to 6. The sizes are
125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray
about 25 cm. But my gel was not properly formed. i will be highly
delighted if you could suggest me something, like the % of agarose I
should use, run time, gel lengh, voltage etc.
Thanks in advance
Rashmi Gupta
PhD Student
AIIMS
New Delhi

Dear Rashmi,

If you (or your PI) insist(s) on agarose, you might need some special
grade suitable for high percentage gels.

Do you have the possibility to run Acrylamide gels instead?
Then you might use silver staining to visualize the DNA.

Best regards,

Wolfgang

Dear Rashmi,

I suggest on you to use MetaPhore agarose, 4.5 % will be very good for small
fragments. Be aware that you need to cook it carefully. Fist use cold buffer
(stored on 4C), add the MetaPhore garose powder little by little while
mixing using magnetic stirrer, let the mix for 15 minutes then cook it in
the microwave starting at low heat then increase the temperature gradually
(it needs 20- 30 min ).

For running the gel, 150 watts for 4-5 hours, do not put stain (Ethedium
bromide) with the gel, just incubate the gel after running for 15-20 min in
a bath of Eth. Brd diluted with distilled water

All the best,
Omar

-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of rashmi
Sent: Monday, August 11, 2008 6:31 PM
To: [Only registered users see links. ]
Cc: [Only registered users see links. ]
Subject: high percentage agarose

Hi there,
I am a PhD student working on Pneumocystis biodiversity. I cam across
your message posted on google groups, regarding high percentage
agarose gels and TBE concentration. One of the objective of my thesis
is to do typing by counting number of 10 base pair repeats in my PCR
products. The number of repeats can vary from 2 to 6. The sizes are
125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray
about 25 cm. But my gel was not properly formed. i will be highly
delighted if you could suggest me something, like the % of agarose I
should use, run time, gel lengh, voltage etc.
Thanks in advance
Rashmi Gupta
PhD Student
AIIMS
New Delhi
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In article <[Only registered users see links. ]>, rashmi <[Only registered users see links. ]> wrote:

This is definitely a job for acrylamide gel. Google a bit and you
shall find.

DK

On Aug 11, 4:21 pm, [Only registered users see links. ] (DK) wrote:

I have done in the pas discrimination of 3 bp 126/129 allele on 6%
agarose. The trick was to boil agarose for long time and to add some
urea in it while is boiling. So if you need to make 100 ml 6% agarose
start with 200 ml H2O +10ml TBE + 6g Agarose, when is boiling add 1-2
g Urea, keep boiling until volume is reduced to 100 ml and immediately
pour without cooling. Don't worry about bubbles, they will come out.
One more thing - run the gel as fast as you can (high voltage /cm) .
Good luck

The opening line always fascinates me! The email was directed to the
methods listserve, and to be sure, there would be someone in this group wo
had posted on some unspecified "google groups"!

Anyway, I suggest a mini acrylamide sequencing set-up. There are many
protocols for that in places as you have mentioned.




--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003

A long time ago I was looking for small differences between fragments and used pre-cast polyacrylamide gels (from Bio-Rad I think), which I stained with a gel-star nucleic acid stain (currently sold by Lonza). Worked really well - Magda

Magda Dunowska, LW (vet), PhD
Senior Lecturer in Veterinary Infectious Diseases (Virology)
Institute of Veterinary, Animal and Biomedical Sciences
Te Kura Mâtauranga Kararehe
Massey University
Palmerston North
New Zealand

Phone : (06) 356-9099 ext 7571
Website : [Only registered users see links. ]
-----Original Message-----
From: [Only registered users see links. ] [mailto:[Only registered users see links. ].indiana.edu] On Behalf Of Dr. Hiranya S. Roychowdhury
Sent: Tuesday, 12 August 2008 9:22 a.m.
To: rashmi
Cc: [Only registered users see links. ]; [Only registered users see links. ]
Subject: Re: high percentage agarose

The opening line always fascinates me! The email was directed to the
methods listserve, and to be sure, there would be someone in this group wo
had posted on some unspecified "google groups"!

Anyway, I suggest a mini acrylamide sequencing set-up. There are many
protocols for that in places as you have mentioned.




--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003

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