Restriction Digest: Partial: Difference between revisions
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==Abstract== | |||
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. | |||
==Materials== | |||
* 10 microcentrifuge tubes | |||
===Reagents=== | |||
* Restriction Endonuclease | |||
* 10X Restriction Endonuclease buffer | |||
* BSA (10 mg/mL) | |||
* Genomic DNA (Concentration > 0.1 μg/μL) | |||
==Procedure== | |||
# 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 **[[Image:Critical step.png]]'''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. | |||
==Acknowledgments== | |||
Acnkowledge any help you had in development, testing, writing this protocol. | |||
==References== | |||
See [[OpenWetWare:Biblio]] for information on how to reference within a wiki. | |||
==Specific Protocols== | |||
Add links to all the OWW protocols that have been used in making the consensus. | |||
==Discussion== | |||
You can [[Talk:{{PAGENAME}}|discuss this protocol]]. | |||
[[Category:Protocol]] | |||
[[Category:In vitro]] | |||
[[Category:DNA]] |
Revision as of 05:17, 18 June 2009
Abstract
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.
Materials
- 10 microcentrifuge tubes
Reagents
- Restriction Endonuclease
- 10X Restriction Endonuclease buffer
- BSA (10 mg/mL)
- Genomic DNA (Concentration > 0.1 μg/μL)
Procedure
- 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.
Acknowledgments
Acnkowledge any help you had in development, testing, writing this protocol.
References
See OpenWetWare:Biblio for information on how to reference within a wiki.
Specific Protocols
Add links to all the OWW protocols that have been used in making the consensus.
Discussion
You can discuss this protocol.