Endy:Colony PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Reaction Mix)
Current revision (04:44, 19 November 2009) (view source)
(BioStream version)
 
(6 intermediate revisions not shown.)
Line 2: Line 2:
==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 reaction:
+
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 (hold)
+
*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]]

Current revision

See Colony PCR for general information about this protocol and other variants

Contents

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

Text Output

Endy:Colony PCR protocol

Source Code

Endy:Colony PCR protocol - source code

Personal tools