User:Anthony Salvagno/Notebook/Research/2010/05/20/Lambda DNA Visualization with QDs
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Lambda DNA Visualization with Quantum Dots
Anthony Salvagno, Steven J Koch
- Lambda DNA is a 48kb linear DNA from lambda phage. It has two complimentary overhangs at the ends of the DNA. Here we produce an attempt at visualization with quantum dots. In order to attach the QDs we perform a Klenow fill-in reaction with biotinylated dCTP in the nucleotide mixture. We use streptavidin conjugated QDs of two colors, 525nm and 625nm from Invitrogen. Current progress will be detailed.
Klenow reaction can be found here. Gel analysis prep can be found here and the results can be found here.
For QD visualization preliminary studies were carried out in glass microfluidics chambers (protocols for construction can be found here). QDs were mixed with lambda DNA in microcentrifuge tubes and allowed to incubate for a short time (specific methods can be found here). 10ul of the mixture was then flowed into the chamber. The sample was then visualized on an Olympus microscope.
The results are inconclusive and will require further analysis. For a while, QDs could not be visualized and then another sample was prepared with just QDs for comparison. The QDs could not be visualized here either. Upon switching back to the DNA/QD sample, something could be seen and this was significantly different than before, but it is not certain what was being seen. THe sample looked like stuck QDs, but there was no blinking. There were also some fluorescent things floating in solution, but it is not certain what those are either. Whatever it was needed to be diluted.
This exact experiment will be replicated, along with a diluted sample for comparison and a sample of just QDs to make sure there is proper visualization.