Can anyone please help me to prepare primer working solution 100ng/ul? As I need 125ng primer concentration for my mutagenesis experiment.
I have 100uM stock (Made by diluting 395.9ug in 310.1 ul sterile water)
[Additional information: Primer is 42 bp long
MW= 12766.46 g/mole
Total nmole= 31 nmole]
I really stucked here I would be grateful if anyone can help me to understand.
Thank you
If you follow the calculation as outlined in the link below:
https://onlineconversion.vbulletin.net/forum/main-forums/convert-and-calculate/14661-convert-from-um-to-ng-ul
you will need to dilute using the ratio of 1:1000.
After you add 310.1 uL of water you have a stock solution of = 395.9/310.1
=1.277ug/uL. i.e 1277 ng/uL. add 85.9 uL more water it will become 1000 ng/uL. after this take 100 uL and add 900 uL of water in it. You will get a stock of 100 ng/uL. Hope this will help you for any doubt feel free to ask.
According to you explanation do you have a concentration of: 1,277 ug/ul (that is equal to 1277 ng/ul)
You need to employ this basic equation:
Initial Volume X Initial Concentration = Final Volume X Final Concentration
310.1 ul X 1277ng/ul = X X 1000ng/ul
395,997 / 1000 = 395,9 ul (It is the final volume, but you have 310,1 so, you need add 85,9 ul of TAE or Nuclease Free water to obtain a concentration of 1000 ng/ul).
Then you can dilute 1:10 to obtain 100 ng/ul solution or 1:100 to 10 ng/ul solution. (It is important because, if your stock is 100 ng/ul and you need 125 ng, you have to take 1,25 ul. be very careful with that).
Everyone has explained very well. Additionally, I think following links are very helpful in calculating accurately.
https://www.graphpad.com/quickcalcs/molarityform.cfm
https://dharmacon.horizondiscovery.com/tools-and-calculators/bioit-beta-tools/nmol-to-ug-calculator/
https://www.physiologyweb.com/calculators/dilution_calculator_molarity_percent.html
https://www.physiologyweb.com/calculators/dilution_calculator_mass_per_volume.html
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