Hi Polina, the simplest solution is, first fix the total DNA amount to be visualized in gel, i.e., 100 ng.

-Then calculate the amount of sample you need for 100 ng, if your concentration is 20 ng/nl, then you need 100ng divided by 20 ng/ul=5ul sample. if you have different concentrations, your sample volume will be different. i.e., 6ul, 7 ul...so on.

-Now add 1ul of 6XLoading dye in each sample (depending on your sample volume you can increase Loading dye amount).

-Add distilled water to make 10 ul total volume of sample (if you have 6 ul DNA, add 1 ul loading dye, and 3 ul of distilled water to make 10 ul solution). You may scaled up like this way for all your samples.

-For gel electrophoresis, DNA Equal Concentration of Samples are important not the Equal Volume of samples.

Best wishes.

Hi Polina, the simplest solution is, first fix the total DNA amount to be visualized in gel, i.e., 100 ng.

-Then calculate the amount of sample you need for 100 ng, if your concentration is 20 ng/nl, then you need 100ng divided by 20 ng/ul=5ul sample. if you have different concentrations, your sample volume will be different. i.e., 6ul, 7 ul...so on.

-Now add 1ul of 6XLoading dye in each sample (depending on your sample volume you can increase Loading dye amount).

-Add distilled water to make 10 ul total volume of sample (if you have 6 ul DNA, add 1 ul loading dye, and 3 ul of distilled water to make 10 ul solution). You may scaled up like this way for all your samples.

-For gel electrophoresis, DNA Equal Concentration of Samples are important not the Equal Volume of samples.

Best wishes.

a generalised answer is to put your data into the equation C1xV1=C2x V2

wher C1 and c2 are the starting and finishing concentrations and V1 and V2 are the initial and final sample volumes. You know c1 and v1 and c2 so you can calculate v2 and make up the difference in the pcr reaction with water as Sharmin says

I would like to point one thing here which is a 6x loading dye amount depends on the final volume of your sample ( in your case DNA and water volumes together). 1ul of 6X dye for each 5ul final volume. Good luck

Please use 10X loading dye (1 ul), 5 ul sample and 4 ul water. in case 100ng /5ul DNA . Adjust accordingly.

What you describe is a10x, not 6x loading buffer. In a total vol of 10μl you can have as much as 9 μl DNA solution. Practically your problem will be extremely low or high DNA concentrations ( which you can adjust by dilution or concentration).

Hi all, you can calculate your DNA final concentration with the formula Cinitial xV?=Cfinal xVfinal. For example, your concentration of DNA in nanodrop is 200ng/μl and you want each samples to be 50ng/ μl in 20μl of volumen:

200ng/ μl x V?= 50ng/ μl x 20 μl you have to add 5 μl of your DNA sample and add 15 μl od destilated water to reach 20 μl final

I Add and excel table for calculate the concentration,

i hope its helps

PD: the concentration of DNA in nanodrop have to be more than the concentration final.

1. Quantification:

H2O or 1X TE solution of nucleic acids in a clean quartz cuvette (Record dilution factor). Zero the spectrophotometer with H2O or 1xTE. Linearity range of the nucleic acids lies between 0.1 and 1.0.

For a 1-cm pathlength, the OD at 260 nm will be 1.0 for the following NA solutions: 50 μg/mL solution of dsDNA, 33 μg/mL solution of ssDNA, 20-30 μg/mL solution of oligonucleotide, 40 μg/mL solution of RNA This is not absolutely correct but can be used for purity data.Pure DNA has an OD260/OD280 ratio of ~1.8; pure RNA has an OD260/OD280 ratio of ~2.0. Low ratios could be caused by protein or phenol contamination. Then, dilution may be done accordingly (as of Rocio Olivera).

Dear Polina,

My experience with NanoDrop is that after dilution you will get "theoretically calculated concentration". We have been preparing a large batch of samples with same concentration of 2 ng/mcl, diluting stock samples correspondingly to dilution calculations. That we checked the final solutions and their concentration was closed to 2 ng/mcl, but not always exact.

So, if you use NanoDrop 2000 or similar measuring the drops, you should mention such aspect in the methods description. If you use cuvettes - follow recommendations from Mohammad.

And one more factor we've noticed is good mixing after dilution as well as pipetting before load.

Good luck!!!