Agarose gel electrophoresis: Difference between revisions

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==Endy Lab==
{{back to protocols}}
Agarose gel electrophoresis is used to separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field (electrophoresis). Shorter molecules move faster and migrate further.


===Pouring agarose gels===
== General Procedure ==
#Cast a gel
#Place it in gel box in running buffer
#Load samples
#Run the gel
#Image the gel


Purpose: To prepare gels for gel electrophoresis.
== Casting Gels ==


====Materials====
[[Image:Agarose gel dyes.jpg|thumb|right|175px|0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes]]
<center>
'''The amount of agarose to use in your gel depends on the DNA in question.  Use the following table as a rough guide:'''


*gel box, gel tray, comb <br> ''all in gel room''
{| border="1" cellpadding="5" cellspacing="0" align="center"
*1% agarose in TAE <br> ''premade, premelted bottles available in Peltier incubator; next room over on the floor''
|+
*[[Ethidium Bromide]] (abbreviated EtBr) <br> ''in gel room, upper right hand shelf''
! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)
*prewarmed glass bottle
|-=
| align="center"|0.5 || align="center"|1 - 30
|-
| align="center"|0.7 || align="center"|0.8 - 12
|-
| align="center"|1.0 || align="center"|0.5 - 10
|-
| align="center"|1.2 || align="center"|0.4 - 7
|-
| align="center"|1.5 || align="center"|0.2 - 3
|}
</center>


====Procedure====
# Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see below). The volume required depends on your gelbox / casting system -- 50mL makes a good, thick gel for a 7x10cm gelbox. 
# Microwave until the agarose is fully melted.  This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results.  As long as you do not burn the agarose and nothing bubbles over, this step is robust.
# Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel).  At this point add your DNA stain, e.g., ethidium bromide.  The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
# While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side.  Make sure it is sealed well or the gel will leak.
# Pour the agarose solution into the taped gelbox.  Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.


#Place gel tray in box to pour. The rubber ends of the tray should form a seal with the sides of the box.
If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
#Place comb of appropriate width, size, and number into niche at end of the tray.
#Prewarm a small 100 ml bottle (preferably with marks on the side indicating volume); setting it in the water bath for a few minutes should do it, or the incubator.
#Pour 35 ml of 1% agarose in 1X TAE for small gels, 70ml for large gels and place into prewarmed bottle. The marks on the side of the bottle should be a sufficient guide.
#Add 0.2 &mu;l of EtBr per 35 ml 1% agarose. '''EtBr is extremely carcinogenic - be careful with it.''' (Wear nitrile gloves; dispose of tips properly).
#Swirl bottle to mix EtBr. Pour the mixture into the tray.
#Let agarose set for ~15 minutes to solidify. After it has set a bit (enough so that you can walk with it), you can set it in the cold room to chill further and solidify completely, saving a few minutes.
#When gel has solidified, remove the comb. Lift the gel box and rotate it 90&deg;, so that the ends of the gel are now exposed to either end of the box.
#Add 1X TAE so that it fills the reservoirs at both ends of the gel and covers the surface of the gel -- the gel should be immersed in 1X TAE. (There are bottles of 1X TAE in the gel room. If they're empty, 1X TAE is above the sink in the main room.)


You're now ready to load DNA samples and run your gel.
== Buffers ==
* [[TAE]] - better resolution of fragments >4kb;
* [[TBE]] - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity;
* [[SB]] - Sodium borate - low heat generation with good resolution. Allows for higher voltage (250-200V) electrophoresis without melting.


===Running agarose gels===
== Loading dyes ==
{| cellpadding=5 style="border: 1px solid #CC9;"
| dye
| 0.5-1.5% agarose
| 2.0-3.0% agarose*
|-
| xylene cyanol
| 10'000-5000 bp
| 750 bp
|-
| bromophenol blue
| 400-500 bp
| 100 bp
|}


==Knight lab==
<nowiki>*</nowiki>sieving agarose


===Casting agarose gels===
for recipes see: [[Agarose gel loading dye]]


We precast our gels.
==Notes==
*If you are getting unexpected bands on your gel you may want to look at the [[Agarose gel electrophoresis/Common issues | common issues in agarose gel electrophoresis page]].
*If you have no experience with gel electrophoresis or are explaining it to someone new, [http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html here] is a cute java demo of what happens.
*Unless your gel box is made from temperature sensitive materials, I've found the cooling step unnecessary, and risky if you get distracted easily.
*Often, the amount of dye you use can be dramatically reduced if you add it directly to your DNA sample and not to the entire gel.  This is the case with SYBR green.


====Materials====
==See also==
* [[Agarose gel loading dye]]
Specific Protocols
* [[Endy:Agarose gel]]
* [[Knight:Agarose gel electrophoresis]]
* [[Alm:Agarose gel electrophoresis]]
* [[Richard Lab:Agarose Gel Electrophoresis]]
[[Agarose_gel_electrophoresis/Common_issues|Common Issues]]


*[[1X TAE]]
==External links==
*[[Ethidium Bromide]] (10 mg/mL stock)
* Wikipedia entry on [http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis agarose gel electrophoresis] [[Image:3stars.png]]
*Agarose
* Colorado state has an excellent [http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html page with detailed instructions and photos] [[Image:3stars.png]]
*Microwave
* [https://www.roche-applied-science.com/PROD_INF/MANUALS/labfaqs/lab_faqs_%2001.pdf Roche's Lab FAQ section on DNA work] [[Image:3stars.png]]
*Stir plate and stir bar (optional)
* Methodbook.net has a nice [http://www.methodbook.net/dna/agarogel.html primer on agarose gel electrophoresis] [[Image:2stars.png]]
* CSH Protocols (~Molecular Cloning) protocol on [http://www.cshprotocols.org/cgi/content/full/2006/2/pdb.prot4020 agarose gel electrophoresis] [[Image:3stars.png]] but restricted access


====Procedure====
[[Category:Protocol]]
 
[[Category:In vitro]]
#Add 300mL 1X TAE to a 500 mL bottle.
[[Category:DNA]]
#Measure out sufficient agarose to cast either a 1% (3 g) or 1.5% (4.5 g) gel.
[[Category:RNA]]
#Add the agarose to the TAE buffer in the 500 mL bottle.
#Swirl to mix.
#Microwave bottle with loosened cap on high until the gel starts to bubble and is transparent.  <br> ''This generally takes just over two minutes for 300 mL.  If you microwave too long, the gel will bubble over causing a big mess and you will need to start over.''
#Remove from microwave and let cool by either sitting on bench top or adding stir bar and placing on stir plate. <br> ''The advantage of the stir plate is that, if you forget about your gel for a while, it is less likely to solidify accidentally. <br> If you are in a hurry, you can place the bottle in a beaker of room temperature water on the stir plate to speed the cooling process significantly.''
#While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.
#Once gel is cooled so that it can be touched comfortably with your gloved hand, add 15 &mu;L Ethidium Bromide (final concentration of 0.5 &mu;g/mL).
#Pour gel into casting trays. <br> ''The height of the gel will depend on how much you wish to load.  Diagnostic gels can be reasonably shallow since typically 10 &mu;L volumes are loaded.  For gel purifications, the gel should be deeper to enable loading of large sample volumes.''
#Let gels sit until they are solidified.  <br> ''Gels are solid when they are cloudy in appearance and firm to the touch.''
#Gels may be used immediately.  Alternatively, gels may be individually sealed in 6 x 10 inch polyethylene bags, labelled with initials, date and percentage and stored at 4 &deg;C.  <br> ''It is a judgement call as to whether a gel is too old to be used.  If it takes on a shrivelled appearance, don't use it.  If there is lots of condensation on the bag, only use it if your intended experiment isn't critical.''
 
===Running agarose gels===
 
====Materials====
 
*[[DNA ladder | Prepared DNA ladder]]
*Precast gel with the appropriate percentage and well size/numbers for your samples (see above)
*[[1X TAE]]
*[[Loading dye]]
*[[Ethidium Bromide]] (10 mg/mL stock)
 
====Procedure====
 
#Take a gel from the 4&deg;C fridge.  <br> ''If the number of gels is getting low, cast more gels as described above.''
#Place your gel in gel box.
#Add 1X TAE buffer to gel box such that buffer just covers the top of the gel.
#Remove comb.
#Add 10 &mu;L ethidium bromide stock solution to the running buffer well near the positive terminal.
#Load 12 &mu;L prepared ladder <br> ''Typically load laddder in left-most lane and sometimes right-most lane as well depending on whether you have the space.''
#Use 2 &mu;L loading dye per 10 &mu;L of sample.
#Load samples left to right. <br> ''The capacity of the 8 well, 1.5mm wide well is approximately 45 &mu;L.  The capacity of the 15 well, 1.5mm well is approximately 15 &mu;L.''
#Place gel box cover on gel box such that your samples will run towards the positive, red electrode.
#Run your gel at ~85 volts for 1 hr 20 mins.  Use the timer to enable automatic shutoff of your gel. <br> ''If you are in a hurry the gel can be run faster at ~95 volts for less than an hour.''
#Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.
 
===Visualizing agarose gels===
 
''Note that this procedure is under development''
 
====Materials====
 
*FluorChem 8800 gel imager
 
====Procedure====
 
#Remove gel from gel box shaking gently to allow residual buffer to fall back into gel box.
#Place in middle of UV box inside gel imager (you can leave the gel in gel tray).
#Close the door and turn on reflective white light button.
#In gel imager software, click the "Acquire" button such that gel displays on screen.
#Adjust gel position on UV box so that the entire gel is within the frame.
#Close the door, turn off reflective white light and turn on transilluminating UV light.
#In gel imager software, click "Acquire image" button to capture gel image to the screen. <br> ''It is occasionally necessary to adjust exposure time to improved image.''
#Increase filtering if bands are difficult to see.
#Annotate gel as necessary.
#Save a copy of gel picture in your user folder.
#Print.
#Remove gel and dispose in ethidium bromide waste container.
#Wipe down UV box if necessary.
 
==Interpreting results==
 
If you are getting unexpected bands on your gel you may want to look at the [[Common Agarose Gel Issues]]

Latest revision as of 05:42, 11 September 2014

back to protocols

Agarose gel electrophoresis is used to separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field (electrophoresis). Shorter molecules move faster and migrate further.

General Procedure

  1. Cast a gel
  2. Place it in gel box in running buffer
  3. Load samples
  4. Run the gel
  5. Image the gel

Casting Gels

0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes

The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:

Agarose Concentration (g/100mL) Optimal DNA Resolution (kb)
0.5 1 - 30
0.7 0.8 - 12
1.0 0.5 - 10
1.2 0.4 - 7
1.5 0.2 - 3
  1. Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see below). The volume required depends on your gelbox / casting system -- 50mL makes a good, thick gel for a 7x10cm gelbox.
  2. Microwave until the agarose is fully melted. This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
  3. Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
  4. While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.
  5. Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.

If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.

Buffers

  • TAE - better resolution of fragments >4kb;
  • TBE - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity;
  • SB - Sodium borate - low heat generation with good resolution. Allows for higher voltage (250-200V) electrophoresis without melting.

Loading dyes

dye 0.5-1.5% agarose 2.0-3.0% agarose*
xylene cyanol 10'000-5000 bp 750 bp
bromophenol blue 400-500 bp 100 bp

*sieving agarose

for recipes see: Agarose gel loading dye

Notes

  • If you are getting unexpected bands on your gel you may want to look at the common issues in agarose gel electrophoresis page.
  • If you have no experience with gel electrophoresis or are explaining it to someone new, here is a cute java demo of what happens.
  • Unless your gel box is made from temperature sensitive materials, I've found the cooling step unnecessary, and risky if you get distracted easily.
  • Often, the amount of dye you use can be dramatically reduced if you add it directly to your DNA sample and not to the entire gel. This is the case with SYBR green.

See also

Specific Protocols

Common Issues

External links

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