Polyacrylamide gel electrophoresis
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.
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.
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
Any equipment used to perform the protocol (link to a method for using them).
1. Place Glass plates in casting apparatus
2. Add together the following to make 5ml of gel (0.75mm spacers)
- 3ml water
- 3ml water
- 1.5ml 29:1 acrylamide solution (40% w/v)
- 500µl10X TBE solution
- 35µl Ammonium Persulfate (10%w/v)
- 2µl TEMED
3. Pipet mix between casting plates using 5ml pipetor
4. Insert comb and allow to cure for 30 minutes
5. Mix the following for all DNA samples including the ladder
1ul SYBR green (1:100 Dilution in DMSO)
2ul Bromophenol Blue
6. Remove comb, insert gel into electrophoresis chamber and add the necessary amounts of 1X TBE
7. Add DNA mix to wells.
8. Apply 80 volts and run for approximately 60 minutes
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
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.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
It might also be good to add an image to show the workflow and timescales for experiment planning.
Acnkowledge any help you had in development, testing, writing this protocol.
See OpenWetWare:Biblio for information on how to reference within a wiki.
Add links to all the OWW protocols that have been used in making the consensus.
You can discuss this protocol.
Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.