Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Molecular Biology Articles and Protocols
Register Search Today's Posts Mark Forums Read

Molecular Biology Articles and Protocols Post Molecular Biology and science Protocols, Reviews, and Articles Forum. You can be the author!


Agarose Gel Electrophoresis and DNA Fragment Purification

Agarose Gel Electrophoresis and DNA Fragment Purification - Molecular Biology Articles and Protocols

Agarose Gel Electrophoresis and DNA Fragment Purification - Post Molecular Biology and science Protocols, Reviews, and Articles Forum. You can be the author!


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-31-2006, 11:17 PM
Pipette Filler
Points: 2,792, Level: 34 Points: 2,792, Level: 34 Points: 2,792, Level: 34
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2006
Posts: 26
Thanks: 0
Thanked 3 Times in 2 Posts
Post Agarose Gel Electrophoresis and DNA Fragment Purification



Supplies:

1. Micropipetter and tips
2. Microcentrifuge and tubes
3. Gel apparatus
4. Power supply for the gel apparatus
5. Polaroid(R) setup (with proper filter - SYBR Green/Gold gel stain photographic filter) and UV light box Teacher's note: UV is dangerous to the skin and eyes, so supervision is very important.
6. Microwave

Procedures:

1. Set up 1 % (w/v) agarose gel:
* Add 0.5 g agarose powder to a flask.
* Add 50 ml TAE buffer to make enough for the gel apparatus.
* Heat (by microwave) briefly to dissolve the powder.Teacher's note: Do not allow it to boil over, and use protection since the flask is hot.
* Cool to 50-60 degrees C.
* Pour into the gel tray.
* Lower the comb (to form wells) gently into the molten gel.
* Allow the gel to solidify in the refrigerator for 15 minutes.Teacher's note: Care should be taken to make sure the gel is leveled while solidifying.
2. Prepare DNA marker:
* Take 3 ul of DNA marker stock solution into a tube.
* Add 22 ul of distilled water.
* Add 5 ul of 6 X loading dye and mix. (observation: contents in the tubes change color from the addition of loading dye.)
3. Add 4 ul of 6 X loading dye into 20 ul of samples for gel electrophoresis. (observation: contents in the tubes change color from the addition of loading dye.)Teacher's note: The amount of DNA used varies with the purpose of the agarose gel electrophoresis.
4. Spin for 2 seconds in a microcentrifuge.
5. Put solidified gel into the gel apparatus.
6. Add TAE buffer to cover the gel and remove the comb.
7. Load 10 ul from each tube into a well.
8. Load 10 ul of DNA marker into a well.
9. Run the gel at 100-150 V for 20 minutes. (observation: dye front moves toward the positive charge.)Teacher's note: High voltage is used here, so supervision is very important. Use 100 V with mini-gel apparatus, and use 150 V with midi-gel apparatus.
10. Staine the gel for 30 minutes with a 1:10,000 dilution of SYBR Green I(R) nucleic acid gel stain.
11. Put on proper face shield and any other necessary gear to protect skin and eyes from UV.
12. Move the gel onto UV light box.
13. Take a Polaroid photo under UV.Teacher's note: Supervision is very important. UV is damaging skin, eyes, and also to the DNA. Turn on UV for brief moments only.
14. If necessary, use a spatula to cut out DNA band(s) of interest under UV, transfer into a tube, and store at -20 degrees C.Teacher's note: Supervision is very important. UV is damaging skin, eyes, and also to the DNA. Turn on UV for brief moments only.
Reply With Quote
Reply

Tags
agarose , dna , electrophoresis , fragment , gel , purification


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump


All times are GMT. The time now is 06:48 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11493 seconds with 15 queries