Lidstrom:Overlap Extension PCR
From OpenWetWare
(Difference between revisions)
m |
(→Sewing PCR Without Primers) |
||
| Line 17: | Line 17: | ||
== Sewing PCR Without Primers == | == Sewing PCR Without Primers == | ||
* use template DNA | * 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 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 31: | Line 31: | ||
* 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. |
== Use Primers on "Sewing PCR" product == | == Use Primers on "Sewing PCR" product == | ||
Revision as of 12:59, 11 October 2012
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
- Two stretches of linear DNA with homologous ends can be combined using this technique.
- Useful paper: Gene splicing and mutagenesis by PCR-driven overlap extension
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.
- 98oC, 30sec
- 98oC, 10sec
- 58oC, 10sec
- 72oC, 30sec/kb
- Repeatsteps 2-4 29x NOTE: Justin Siegel/Janet Matsen only do 9x
- 72oC, 5min
- 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.
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.
