Polyacrylamide gel electrophoresis: Difference between revisions

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This procedure is useful for the separation of small pieces of DNA, small quantities of DNA, or applications where higher resolution than can be achieved with agarose gel electrophoresis is needed.  Because it is very difficult to remove DNA from polyacrylamide gel, it is advised that this protocol should only be used for diagnostic purposes and not for size separation.
This procedure is useful for the separation of small pieces of DNA, small quantities of DNA, or applications where higher resolution than can be achieved with agarose gel electrophoresis is needed.  Because it is very difficult to remove DNA from polyacrylamide gel, it is advised that this protocol should only be used for diagnostic purposes and not for size separation of useful fragments.


==Materials==
==Materials==
List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc.  Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
===Reagents===
===Reagents===
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
*29:1 Acrylamide Solution
*10X TBE buffer
*Ammonium Persulfate
*TEMED
*DNA solution
*[[SYBR Green II|SYBR green]]
*Bromophenol Blue
===Equipment===
===Equipment===
Any equipment used to perform the protocol (link to a method for using them).
*Vertical electrophoresis chamber
*Glass plates w/ spacers (which fit the chamber)
*Casting holder
*Pipettors


==Procedure==
==Procedure==
==Procedure==
1. Place Glass plates in casting apparatus<br>
1. Place Glass plates in casting apparatus<br>
2. Add together the following to make 5ml of gel (0.75mm spacers)<br>
2. Add together the following to make 5ml of gel (0.75mm spacers)<br>
:*3ml water<br>
:*500µl 10X TBE solution<br>
*1.5ml 29:1 acrylamide solution (40% w/v)<br>
:*35µl Ammonium Persulfate (10%w/v)<br>
*500µl10X TBE solution<br>
:*X mL 29:1 acrylamide solution (See the table under "notes" to determine the desired acrylamide concentration)<br>
*35µl Ammonium Persulfate (10%w/v)<br>
:*Y mL water (To make 5 mL)<br>
*2µl TEMED<br>  
:*2µl TEMED<br>  
3. Pipet mix between casting plates using 5ml pipetor<br>
3. Pipet the acrylamide solution between the casting plates using a 5ml pipetor.<br>
4. Insert comb and allow to cure for 30 minutes<br>
4. Insert comb into the top of the gel and allow it to cure vertically for approximately 30 minutes.<br>
5. Mix the following for all DNA samples including the ladder<br>
5. Combine the following for all DNA samples including the ladder:<br>
10 DNA<br>
:*10μL DNA solution<br>
1ul SYBR green  (1:100 Dilution in DMSO)<br>
:*1μL SYBR green  (100X Dilution in DMSO)<br>
2ul Bromophenol Blue<br>
:*1μL Bromophenol Blue<br>
6. Insert the gel into the electrophoresis chamber allong with the buffer dam.
6. Remove comb, insert gel into electrophoresis chamber and add the necessary amounts of 1X TBE<br>
:*Make sure both the gel and the buffer dam seal.
7. Add DNA mix to wells. <br>
:*The wells on the gel should face the inside.
8. Apply 80 volts and run for approximately 60 minutes<br>
7. Add 1X TBE to the space between the gel and tue buffer dam until the TBE fills the wells in the gel.<br>
 
:*No TBE should leak into the space outside of this chamber.
==Critical steps==
8. Add 1X TBE to the outer chamber to the specified fill level.<br>
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
9. Add DNA mix to wells. <br>
 
10. Apply 80 volts and run for approximately 60 minutes.<br>
==Troubleshooting==
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.


==Notes==
==Notes==
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br>
[[Image:Vertical_electrophoresis.JPG|300px|right]]
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>


It might also be good to add an image to show the workflow and timescales for experiment planning.
*It is important to get the fill level right in the electrophoresis apparatus (please see the figure to the right).
*For thicker gels (bigger wells) more gel will need to be made.  The amount of gel necessary can be calculated volumetrically.
*The voltage applied to the gel will vary according the the tube of gel you cast.  The specified voltage is 1-8 volts/cm.  Centimeters in this case specifies the length of the gel from top to bottom (i.e the direction the DNA will travel). 
*To determine the final acrylamide concentration for your application use the following table.  For example: if I had a 40% acrylamide stock solution and I wanted 5mL of gel with a final concentration of 12% acrylamide, then I would add 5ml*(12%/40%)=1.5mL of acrylamide solution to make my gel.


==Acknowledgments==
<center>
Acnkowledge any help you had in development, testing, writing this protocol.
{| border="1" cellpadding="5" cellspacing="0" align="center"
|+ '''Choosing an Acrylamide Concentration'''
! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)
|-
| align="center"|3.5 || align="center"|1000-2000
|-
| align="center"|5.0 || align="center"|80-500
|-
| align="center"|8.0|| align="center"|60-400
|-
| align="center"|12.0 || align="center"|40-200
|-
| align="center"|15.0 || align="center"|25-150
|-
| align="center"|20.0 || align="center"|6-100
|}
</center>


==References==
==References==
See [[OpenWetWare:Biblio]] for information on how to reference within a wiki.
*Sambrook J, Russell DW, Cold Spring Harbor L: Molecular cloning : a laboratory manual / Joseph Sambrook, David W. Russell. Cold Spring Harbor, N.Y. :: Cold Spring Harbor Laboratory; 2001.
 
==Specific Protocols==
Add links to all the OWW protocols that have been used in making the consensus.


==Discussion==
==Discussion==
You can [[Talk:{{PAGENAME}}|discuss this protocol]].  
You can [[Talk:{{PAGENAME}}|discuss this protocol]].  


Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.


[[Category:Protocol]]
[[Category:Protocol]]
[[Category:DNA]]
[[Category:In vitro]]

Latest revision as of 12:02, 29 March 2011

This procedure is useful for the separation of small pieces of DNA, small quantities of DNA, or applications where higher resolution than can be achieved with agarose gel electrophoresis is needed. Because it is very difficult to remove DNA from polyacrylamide gel, it is advised that this protocol should only be used for diagnostic purposes and not for size separation of useful fragments.

Materials

Reagents

  • 29:1 Acrylamide Solution
  • 10X TBE buffer
  • Ammonium Persulfate
  • TEMED
  • DNA solution
  • SYBR green
  • Bromophenol Blue

Equipment

  • Vertical electrophoresis chamber
  • Glass plates w/ spacers (which fit the chamber)
  • Casting holder
  • Pipettors

Procedure

1. Place Glass plates in casting apparatus
2. Add together the following to make 5ml of gel (0.75mm spacers)

  • 500µl 10X TBE solution
  • 35µl Ammonium Persulfate (10%w/v)
  • X mL 29:1 acrylamide solution (See the table under "notes" to determine the desired acrylamide concentration)
  • Y mL water (To make 5 mL)
  • 2µl TEMED

3. Pipet the acrylamide solution between the casting plates using a 5ml pipetor.
4. Insert comb into the top of the gel and allow it to cure vertically for approximately 30 minutes.
5. Combine the following for all DNA samples including the ladder:

  • 10μL DNA solution
  • 1μL SYBR green (100X Dilution in DMSO)
  • 1μL Bromophenol Blue

6. Insert the gel into the electrophoresis chamber allong with the buffer dam.

  • Make sure both the gel and the buffer dam seal.
  • The wells on the gel should face the inside.

7. Add 1X TBE to the space between the gel and tue buffer dam until the TBE fills the wells in the gel.

  • No TBE should leak into the space outside of this chamber.

8. Add 1X TBE to the outer chamber to the specified fill level.
9. Add DNA mix to wells.
10. Apply 80 volts and run for approximately 60 minutes.

Notes

  • It is important to get the fill level right in the electrophoresis apparatus (please see the figure to the right).
  • For thicker gels (bigger wells) more gel will need to be made. The amount of gel necessary can be calculated volumetrically.
  • The voltage applied to the gel will vary according the the tube of gel you cast. The specified voltage is 1-8 volts/cm. Centimeters in this case specifies the length of the gel from top to bottom (i.e the direction the DNA will travel).
  • To determine the final acrylamide concentration for your application use the following table. For example: if I had a 40% acrylamide stock solution and I wanted 5mL of gel with a final concentration of 12% acrylamide, then I would add 5ml*(12%/40%)=1.5mL of acrylamide solution to make my gel.
Choosing an Acrylamide Concentration
Acrylamide Concentration(%) Optimal DNA Resolution (bp)
3.5 1000-2000
5.0 80-500
8.0 60-400
12.0 40-200
15.0 25-150
20.0 6-100

References

  • Sambrook J, Russell DW, Cold Spring Harbor L: Molecular cloning : a laboratory manual / Joseph Sambrook, David W. Russell. Cold Spring Harbor, N.Y. :: Cold Spring Harbor Laboratory; 2001.

Discussion

You can discuss this protocol.

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