My DNA is about 500 BP and that is about 100 ng/5ul for the DNA mass according to the SMOBIO DNA Ladder.
I need a concentration of 2ng/ul for sequencing, and it also says I need 5 ng and 2.5 ul for the total amount for each 20 ul cycle sequence reaction.
How do I do this? Do I know my concentration for my DNA from the ladder or do I need to use the nanodrop? How to I dilute to the right concentration?
Dear Amanda Hill
How you determine concentration of your DNA with ladder?
It was first time I heard that and I am very interesting to know.
You dilute your DNA using the c1v1 = c2v2 formula
c1 is starting concentration
v1 is starting volume
c2 is final concentration
v2 is final volume
I would usually dilute PCR in molecular grade water.
Estimating brightness from a gel will get you "close enough" for sequencing reactions. Or you could use a nano drop if you want a more precise measurement.
Hello. I would not rely on a DNA mass ladder for an accurate or precise DNA concentration. A nanodrop or equivalent instrument is better.
To answer Alireza's question about how to determine DNA concentration from a ladder, there exist DNA mass ladders where you load a specific volume of the ladder with a loading dye and you compare the brightness of you sample band against the brightness of the bands in the ladder that have a known DNA concentration.
Dera Amanda Hill ,
Greetings, after DNA isolation you need check the quality of your DNA using agrose gel using DNA ladder to determine DNA concentration or using spectrometer. Depend on the band quality and its strong on the gel you could try dilute you DNA into 1:5 or 1:10
with best regards
khaled
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