Restriction Digest: Partial
This protocol can be used to generate genomic DNA fragments of different sizes using a common cutter. This is useful for many screening/probing processes.
- 10 microcentrifuge tubes
- Restriction Endonuclease
- 10X Restriction Endonuclease buffer
- BSA (10 mg/mL)
- Genomic DNA (Concentration > 0.1 μg/μL)
- Label the microcentrifuge tubes 1 - 10
- Mix together the following and invert to mix:
- 180μL Genomic DNA
- 20μL 10X Buffer
- 2μL BSA
- Dispense 40μL of this mix into tube 1 and 20μL into tubes 2 - 10 ** From this point keep everything on ice!!!
- Add 1μL of restriction-endonuclease to tube 1 and invert to mix.
- Transfer 20μL from tube 1 to tube 2 and invert to mix.
- Repeat the last step transfering 20μL from the preceding tube to the succeeding tube mixing with every transfer.
- Finally remove 20μL from tube 10
- Incubate all 10 tubes for 1 hour at 37°C.
- Finish the digest by submerging all 10 tubes in an 70°C water bath for 20 minutes.
- Run the samples on a gel and choose the size which best fits your application.
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.