Difference between revisions of "Restriction Digest: Partial"

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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 **[[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.
Acnkowledge any help you had in development, testing, writing this protocol.
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==Specific Protocols==
Add links to all the OWW protocols that have been used in making the consensus.
You can [[Talk:{{PAGENAME}}|discuss this protocol]].
[[Category:In vitro]]

Revision as of 04:15, 18 June 2009