Endy:Colony PCR: Difference between revisions
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==Protocol== | ==Protocol== | ||
*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water. | *Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water. | ||
*store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an [[Index plate]]. | |||
===Reaction Mix=== | ===Reaction Mix=== | ||
Use the following reaction mix for each PCR | Use the following reaction mix for each PCR: | ||
*1 μl 10x Thermo polymerase buffer | *1 μl 10x Thermo polymerase buffer | ||
*1 μl 10x dNTPs (10x = 2.5 mM each dNTP) | *1 μl 10x dNTPs (10x = 2.5 mM each dNTP) | ||
| Line 17: | Line 18: | ||
*Cycle 35 times: | *Cycle 35 times: | ||
**95 C for 30 s (melting) | **95 C for 30 s (melting) | ||
**53 C for 30 s (annealing) | **53 C (or whatever temperature is appropriate) for 30 s (annealing) | ||
**72 C for X s (elongation) | **72 C for X s (elongation) | ||
*72 C for 10 minutes (final elongation) | *72 C for 10 minutes (final elongation) | ||
*4 C forever | *4 C forever | ||
*For long amplicons, X = 1 minute + 2.5 s per 100bp | *For long amplicons, X = 1 minute + 2.5 s per 100bp | ||
*For shorter amplicons, under ~1kb, this can be shortened judiciously. | *For shorter amplicons, under ~1kb, this can be shortened judiciously. | ||
==BioCoder version== | |||
Following is the Endy:Colony PCR protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[Endy:Colony PCR protocol]] | |||
====Source Code==== | |||
[[Endy:Colony PCR protocol - source code]] | |||
[[Category:Escherichia coli]] [[Category:Protocol]] | |||
Latest revision as of 02:44, 19 November 2009
See Colony PCR for general information about this protocol and other variants
Protocol
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
- store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.
Reaction Mix
Use the following reaction mix for each PCR:
- 1 μl 10x Thermo polymerase buffer
- 1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
- 0.15 μl 40 μM FWD primer
- 0.15 μl 40 μM REV primer
- 0.1 μl Polymerase (taq or vent)
- 6.6 μl H2O
- 1.0 μl template suspension
PCR protocol
- 95 C for 6 minutes (disrupt cells, separate DNA)
- Cycle 35 times:
- 95 C for 30 s (melting)
- 53 C (or whatever temperature is appropriate) for 30 s (annealing)
- 72 C for X s (elongation)
- 72 C for 10 minutes (final elongation)
- 4 C forever
- For long amplicons, X = 1 minute + 2.5 s per 100bp
- For shorter amplicons, under ~1kb, this can be shortened judiciously.
BioCoder version
Following is the Endy:Colony PCR protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
