Week 9. Semester 3. Preparation for Sequencing

On October 06th the DNA samples extracted from the agarose gels were taken to ASU for sequencing in order to compare the nucleotide sequence of the samples with the databases available online. The protocol for the preparation of samples follows:

DNA Sequencing (11-06-2015)

Utilizing a NanoDrop to quantify the mass of DNA in each tube, the mass for each sample was recorded in ng/mL.

First the NanoDrop was blanked with 1µL of sterile water. Only one blank was necessary for all the samples.

The apparatus was wiped with a KimWipe in between the blank and each sample.

1µL of each sample and positive control (purified from agarose gels) was used per sample.

Once the mass was recorded, the tubes were prepared as follows for sequencing:

Each sample had to contain at least 20ng per tube. The smallest mass recorded was ≈5ng/mL. Therefore, 4µL of each sample and controls were added to each labeled tube along with another volume of forward primer. Depending on the sample type, mip or 16s primer was added. 

For reference, I have attached a picture of a similar sequencing machine to the one the team got briefed on last Friday.

Photo courtesy of: ASU Biodesign Institute. Available from: http://cpdlab.biodesign.asu.edu/images/3730.jpg