Dear researcher,
After amplifying my target gene (1900bp) into the required cDNA by PCR,>cut specific gel bands and purify the gel and the concentration was 30ng/ul. Next, performed A-Tailing according to the Kit protocol (Tiangen#RT121221); add 15ul (30ul/ul gel-purified product) + 4ul Tailing A reaction Buffer + 1ul Taq DNA polymerase = 20ul.
For the next pMD™19-T vector cloning kit (TAKARA# code number 6013), the solution needs to be prepared by inserting 0.1 pmol~0.3 pmol of DNA,
1- How to convert ng/ul concentration to pmol/ul?
2- How to calculate the amount of insert DNA from the above?
You need to convert ng to pmol, for what you need toknow the length of the DNA molecules (in basepairs). One pmol basepairs has a mass of 660 pg (=0.66 ng).
(the average molecular mass of DNA is 660 g/(mol x bp))
You can easily convert from ng/μl to nM:
(concenration in ng/ul) / (660 g/mol X average library size in bp) * 106 = concentration in nM.
The average library size you can determine by running it on an Agilent Technologies 2100 or 4200 Bioanalyzer.
Hafiz Muhammad Rizwan Here is the calculator. You can plug in your data to get the result: https://www.promega.com/resources/tools/biomath/
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