Mike Barnkob:Protocols/Bacterial/Diagnostic Restriction Digest: Difference between revisions
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#* For Addgene plasmids, use their suggested enzymes and compare | #* For Addgene plasmids, use their suggested enzymes and compare | ||
#* Use NEBs [https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder Double Digest Finder] to find the correct reaction buffer and if BSA is recommended or not. | #* Use NEBs [https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder Double Digest Finder] to find the correct reaction buffer and if BSA is recommended or not. | ||
#* For each plasmid, prepare | #* For each plasmid, prepare control samples: one without any restriction enzymes (uncut) and one without any DNA. | ||
# Prepare reaction mix in 1.5 mL Eppendort tube: | # Prepare reaction mix in 1.5 mL Eppendort tube: | ||
#* 2 μg DNA | #* 1-2 '''μg''' of DNA | ||
#* 1 μL of each restriction enzyme <br />'''Note:''' Keep all enzymes on ice or ice-block all the time! | #* 1 μL of each restriction enzyme <br />'''Note:''' Keep all enzymes on ice or ice-block all the time! | ||
#* 3 μL of 10x reaction buffer | #* 3 μL of 10x reaction buffer | ||
#* 3 μL of BSA if recommended | #* 3 μL of BSA if recommended | ||
#* Bring total volume to 30 μL with dH20. | #* Bring total volume to 30 μL with dH20. Usually around: | ||
#*# Uncut samples: add 25 μL | #*# Uncut samples: add 25 μL | ||
#*# Without DNA: add 26 μL | |||
#*# With 1 RE: 25 μL | |||
#*# With 2 RE: 24 μL | |||
#* Mix gently | #* Mix gently | ||
# Incubate tube in 37°C water bath for 1 hour. | # Incubate tube in 37°C water bath for 1 hour. | ||
# Make gel when digestion is running. | # Make gel when digestion is running. | ||
# Inactivate reaction by incubating tube on 70°C heat-block for 15 min. | # Inactivate reaction by incubating tube on 70°C heat-block for 15 min. | ||
# Run reaction on gel electrophoresis: | # Run reaction on gel electrophoresis: 40 min at 100 V with 5 μL Loading Buffer. | ||
===References=== | ===References=== | ||
* http://www.addgene.org/recipient-instructions/diagnostic-digest/ | * http://www.addgene.org/recipient-instructions/diagnostic-digest/ | ||
Latest revision as of 02:53, 28 January 2015
Diagnostic Restriction Digest
Diagnostic restriction digest to verify plasmid identity.
Reagents
- Restriction enzymes
- DNA ladder
- Gel
Protocol
- Select restriction enzymes and correct buffer to digest plasmid
- For Addgene plasmids, use their suggested enzymes and compare
- Use NEBs Double Digest Finder to find the correct reaction buffer and if BSA is recommended or not.
- For each plasmid, prepare control samples: one without any restriction enzymes (uncut) and one without any DNA.
- Prepare reaction mix in 1.5 mL Eppendort tube:
- 1-2 μg of DNA
- 1 μL of each restriction enzyme
Note: Keep all enzymes on ice or ice-block all the time! - 3 μL of 10x reaction buffer
- 3 μL of BSA if recommended
- Bring total volume to 30 μL with dH20. Usually around:
- Uncut samples: add 25 μL
- Without DNA: add 26 μL
- With 1 RE: 25 μL
- With 2 RE: 24 μL
- Mix gently
- Incubate tube in 37°C water bath for 1 hour.
- Make gel when digestion is running.
- Inactivate reaction by incubating tube on 70°C heat-block for 15 min.
- Run reaction on gel electrophoresis: 40 min at 100 V with 5 μL Loading Buffer.
