Hello,
I'm interested in optimizing the cost-efficiency of the purification of PCR products from macro fungi for Sanger sequencing using Exo+Sap mixture.
My Exo+Sap product protocol suggests using 2 ul of undiluted Exo+Sap mixture for 4 ul of PCR product. Could I do 1:5 or 1:10 dilutions of my stock Exo+Sap?
If so, what would be the correct proportion of diluted Exo+Sap mixture to PCR product?
Paul Rutland
Hello, Paul. Thank you for your answer. My product already has the two enzymes mixed.
I use the pre-mixed ExoSAP-it reagent also.
10 microliters PCR sample + 1 microliter ExoSAP-it works just fine, so you can "stretch" the reagent to get more samples per tube.
If the issue is that you cannot reliably measure 1-2 microliters, then you can mix the ExoSAP-it with water as long as you use it right away.
e.g. 5 microliters PCR + 5 microliters (1 ExoSAP-it + 4 water)
Calculate how many reactions you'll be cleaning and mix the ExoSAP-it + water. Vortex to make sure it's really well-mixed, then use.
If you are using strip tubes or 96-well plates, portion out the ExoSAP-it into a PCR strip tube & use a multi-channel
Good luck!
You would get some idea of how little exo you can use by taking the definition of 1u of exo in terms of the amount of ssDNA it nucleases and apply that to the amount of primer in 4ul of your reaction and possibly by increasing the incubation time of the exo reaction to degrade more primer. The sap is less important in the reacton as all of the reagents needed for the sequencing reaction are supplied in excess in the sequencing mix
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