Polyacrylamide gel electrophoresis

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(Critical steps)
(Procedure)
Line 24: Line 24:
:*Y mL water (To make 5 mL)<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>
:*10μL DNA solution<br>
:*10μL DNA solution<br>
:*1μL SYBR green  (100X Dilution in DMSO)<br>
:*1μL SYBR green  (100X Dilution in DMSO)<br>
:*1μL Bromophenol Blue<br>
:*1μL Bromophenol Blue<br>
-
6. Remove comb, insert gel into electrophoresis chamber and add the necessary amounts of 1X TBE<br>
+
6. Insert the gel into the electrophoresis chamber allong with the buffer dam
-
7. Add DNA mix to wells. <br>
+
:*Make sure both the gel and the buffer dam seal
-
8. Apply 80 volts and run for approximately 60 minutes<br>
+
:*The wells on the gel should face the inside
-
 
+
7. add the necessary amounts of 1X TBE<br>
-
 
+
8. Add DNA mix to wells. <br>
 +
9. Apply 80 volts and run for approximately 60 minutes<br>
==Troubleshooting==
==Troubleshooting==

Revision as of 15:02, 17 June 2009

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.

Contents

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 the necessary amounts of 1X TBE
8. Add DNA mix to wells.
9. Apply 80 volts and run for approximately 60 minutes

Troubleshooting

Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.

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 *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
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References

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Specific Protocols

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