DNA Cleanup Protocol on Left and Right Fragments

     This week during lab, we were working on cleaning up left and right fragments. We are cleaning the sample of everything except the DNA that we want. The protocol I learned for this cleanup is as follows:

We diluted the left and right samples with the DNA Cleanup Binding Buffer according to a 5:1 ratio recommended for fragments. We then loaded sample into a column that was within a collection tube and spun for 1 min at 13,000 rpm. We then proceeded to discard the flow through. After we re-inserted the column in another collection tube, adding 200µL of DNA Wash Buffer and spun again for 1 minute. We repeated the last step once more before transferring the column into a 1.5µL microfuge tube. Finally we added 10µL of DNA Elution Buffer to our samples, ensuring that its going into the center of the matrix. Then, we waited for 1 minute before proceeding to spin it for another 1 minute at 13,000 rpm. After our first elution cycle, we put the samples on the nano drop, the results are below.

Left Fragment- 186.5 ng/µL 1.82 A260/A280 2.01 A260/A230

Right Fragment- 245.5 ng/µL 1.91 A260/A280 2.18 A260/A230

As these numbers were almost perfect, we saved this flow through of the sample, then repeated the Elution Buffer step. We added another 10µL of the Elution Buffer to our matrix, waited for 1 minute, the spun again. The results for our second round are below.

Left Fragment 2- 14.0 ng/µL 1.69 A260/A280 1.57 A260/A230

Right Fragment 2- 24.5 ng/ml 1.80 A260/A280 2.14 A260/A230