Lidstrom:Overlap Extension PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Use Primers on Gel-Purified "Sewing PCR" product)
Current revision (16:55, 8 March 2013) (view source)
(If you are sewing larger pieces)
 
(5 intermediate revisions not shown.)
Line 4: Line 4:
This method can be used for cloning.  It can also be used to assemble neighboring fragments in Gibson into one piece.  
This method can be used for cloning.  It can also be used to assemble neighboring fragments in Gibson into one piece.  
 +
 +
== The Big Picture ==
 +
* Two stretches of linear DNA with homologous ends can be combined using this technique.
 +
* Useful papers:
 +
** [http://www.ncbi.nlm.nih.gov/pubmed/17446874 Gene splicing and mutagenesis by PCR-driven overlap extension]
 +
** For longer fragments: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373371/?tool=pmcentrez&rendertype=abstract Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously]
== Obtain DNA ==
== Obtain DNA ==
Line 13: Line 19:
== Sewing PCR Without Primers ==
== Sewing PCR Without Primers ==
* use template DNA  
* use template DNA  
-
** Concentration?
+
** Use ~ 50 - 125 ng of each piece per 50 uL.  Each reaction should be 10 - 15 uL: do many so you can test a range of T<sub>anneal</sub> and % DMSO
* Rxn concentrations
* Rxn concentrations
** Use Phusion buffer & standard Phusion dNTP concentration
** Use Phusion buffer & standard Phusion dNTP concentration
-
** Set up an array of annealing temps and % DMSO.  
+
** Set up an array of annealing temps and % DMSO to identify conditions that yield product and minimize unspecific bands.  You can try 55<sup>o</sup>C, 60<sup>o</sup>C, 65<sup>o</sup>C, 70<sup>o</sup>C and 0, 5, & 10% DMSO at each of those temps to cover a very broad range of reaction conditions.  
*Thermocycling: Use Phusion polymerase with default concentrations.  
*Thermocycling: Use Phusion polymerase with default concentrations.  
#98<sup>o</sup>C, 30sec
#98<sup>o</sup>C, 30sec
Line 27: Line 33:
* You can try to run this product on a gel, but it is possible you won't see anything.  (Depends on template DNA concentration in beginning.)
* You can try to run this product on a gel, but it is possible you won't see anything.  (Depends on template DNA concentration in beginning.)
* You can also do the next PCR step without examining on a gel.   
* You can also do the next PCR step without examining on a gel.   
-
** Janet recommends proceeding to this step unless you have problems.  If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.
+
** Janet recommends proceeding to this step unless you have problems.  If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.
 +
 
 +
== If you are sewing larger pieces ==
 +
* The paper ** For longer fragments: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373371/?tool=pmcentrez&rendertype=abstract Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously] is key reading.
 +
* Key points:
 +
** use high (68oC) annealing temps
 +
** don't gel purify PCR products you plan to sew
 +
*** Their experience lead them to believe this is a deal breaker; they rationalized it by DNA damage cause by ethidium bromide and the UV exposure.
 +
** You may need nested primers to amplify the product in the + primer reaction.
 +
*** This is bad news if you are trying to sew Gibson assembly pieces together.
== Use Primers on "Sewing PCR" product ==
== Use Primers on "Sewing PCR" product ==

Current revision

Back to Protocols

Janet is going to write up & illustrate some nice graphics for this! Stay tuned.

This method can be used for cloning. It can also be used to assemble neighboring fragments in Gibson into one piece.

Contents

The Big Picture

Obtain DNA

  • PCR with primers that yield overlapping ends.
    • How much overlap?
  • Gel purify
    • Can sometimes only do a PCR cleanup if your bands are SUPER clean. You will get higher yield if you don't use a gel.

Sewing PCR Without Primers

  • use template DNA
    • Use ~ 50 - 125 ng of each piece per 50 uL. Each reaction should be 10 - 15 uL: do many so you can test a range of Tanneal and % DMSO
  • Rxn concentrations
    • Use Phusion buffer & standard Phusion dNTP concentration
    • Set up an array of annealing temps and % DMSO to identify conditions that yield product and minimize unspecific bands. You can try 55oC, 60oC, 65oC, 70oC and 0, 5, & 10% DMSO at each of those temps to cover a very broad range of reaction conditions.
  • Thermocycling: Use Phusion polymerase with default concentrations.
  1. 98oC, 30sec
  2. 98oC, 10sec
  3. 58oC, 10sec
  4. 72oC, 30sec/kb
  5. Repeatsteps 2-4 29x NOTE: Justin Siegel/Janet Matsen only do 9x
  6. 72oC, 5min
  7. 4oC, forever
  • You can try to run this product on a gel, but it is possible you won't see anything. (Depends on template DNA concentration in beginning.)
  • You can also do the next PCR step without examining on a gel.
    • Janet recommends proceeding to this step unless you have problems. If you don't get decent bands for the next step, run the products from this step on a gel and consider gel purifying them.

If you are sewing larger pieces

  • The paper ** For longer fragments: Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously is key reading.
  • Key points:
    • use high (68oC) annealing temps
    • don't gel purify PCR products you plan to sew
      • Their experience lead them to believe this is a deal breaker; they rationalized it by DNA damage cause by ethidium bromide and the UV exposure.
    • You may need nested primers to amplify the product in the + primer reaction.
      • This is bad news if you are trying to sew Gibson assembly pieces together.

Use Primers on "Sewing PCR" product

  • optional: gel purify the product from the 1st rxn.
    • can run a few uL on a gel to see if non-specific products formed.

Extra Notes

  • Be aware that overloading an agarose gel leads to warped rates of migration through the gel. Include small not-overloaded lanes next to the ladder when running purification gels.
include small not-overloaded lanes when running purification gels
include small not-overloaded lanes when running purification gels
Personal tools