Lidstrom:Overlap Extension PCR
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** 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 | + | == 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 == | == 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. | * 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. | ||
[[image:janet purification gel.jpg|thumb|center|include small not-overloaded lanes when running purification gels]] | [[image:janet purification gel.jpg|thumb|center|include small not-overloaded lanes when running purification gels]] | ||
Revision as of 13:15, 2 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 |
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
- Concentration?
- Rxn concentrations
- Use Phusion buffer & standard Phusion dNTP concentration
- Set up an array of annealing temps and % DMSO.
- 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.
