I want to carry out a single RE digestion using Thermo scientific's NcoI. The reaction protocol requires a DNA concentration of 1ug/uL in a 20uL reaction. My DNA concentration is 1ug in 21.3ul. How do I go about this?
Hi there,
I doubt Thermo is advising to have 1µg/µL of plasmid in the reaction mix. I guess it's 1µg DNA in 20µL. So what you have to do is just to scale up the reaction to let's say 25µL by adding 2.5µL of 10xdigest buffer and 1.3µL of NcoI stock solution and incubate for the indicated time.
Hi there,
I doubt Thermo is advising to have 1µg/µL of plasmid in the reaction mix. I guess it's 1µg DNA in 20µL. So what you have to do is just to scale up the reaction to let's say 25µL by adding 2.5µL of 10xdigest buffer and 1.3µL of NcoI stock solution and incubate for the indicated time.
Hi Nathaniel,
you could also concentrate your DNA by evaporating some water in a speed-vacuum, taking care of quality of your sample.
fred
Go back and check the kit directions. It will tell you a maximum recommended amount of DNA in µg. You can use less DNA than that. Keep in mind that the total reaction volume includes the buffer, DNA, enzyme and water (if needed). You can set up larger reaction volumes and you can omit the water if your DNA is dilute enough.
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