Question

1. You have prepared some plasmid DNA in lab and need to know its concentration so you can load 1 ug on a gel. You dilute 1 uL with 99 uL of water and the resulting sample has an absorbance of 0.075. What volume of the original sample do you need to load onto the gel? (Show math for partial credit and make sure you are answering the question I am actually asking.)
   

Answer 1

When it comes to gel electrophoresis, 1 microgram DNA is very huge quantity of DNA and band will be not properly visible. So, when you dilute it i.e. 1 microlitre + 99 microlitre water and take absorbance using nanodrop, the OD comes to be 0.075 i.e. 0.075 ng/microlitre.

A sample is 100 times diluted ==> Absorbance 0.075 * 100 ====> 7.5 ng/microlitre is the initial concentration of DNA

On agarose gel, you can load 500 ng of DNA and band will be perfectly visible. 500 ng means approx 70 microlitre of the sample.

Also, run the gel at lower voltage i.e.100V if you face similar propblems. Lower voltage helps in proper separation of band inside the gel.

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