As suggested by the formula by Promega's pGEM-T Easy protocol,
(ng of vector x kb size of insert)/kb size of vector x insert:vector molar ratio= ng of insert
using example given by protocol, vector is 3kb and supplied at 50 ng/ul, using ratio 3:1, pcr product 0.5 kb.
hence,
(50 ng vector x 0.5kb insert)/3.0 kb vector x 3/1 = 25 ng insert(PCR product)
My question is,
How do I convert the "ng" to "ul"? I have struggled in the past understanding this conversion.
Can someone kindly teach me the steps to calculate how much in uL of PCR product is equivalent to 25ng ?
Thank you in advance.
The volume depends on the concentration of the insert (PCR product). If your insert is at 25 ng/µL, then to get 25 ng you need to use 1 µL. If your insert is at 50 ng/µL, then to get 25 ng you need to use 0.5 µL. Etc
To measure the concentration of your insert, I would first do a column clean up step after the PCR to remove any excess primers and leftover dNTPs. Then you can quantify the cleaned up PCR product using Qubit or Nanodrop or any other method for quantifying DNA.
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