DNA

Purification

For crystallography, we need DNA that is ultra-pure. When DNA is ordered from a synthesis company, there is a certain level of purity obtained. We can order the DNA very pure but the price sky rockets. Instead, our experimental team collaborated with Dr. Shing Ho, a DNA crystallography lab at CSU, to purify the DNA for our project.

Since this is the Ho Lab’s protocol, we will just share the gist with you.

First, we order the DNA single stranded with the trityl group remaining on the DNA.

This will make sense if you follow the link to find out how most DNA is synthesized when you order it from a company, like IDT. Also, here’s a super cool paper showing all the ways DNA can be synthesized.

Second, we can use reverse phase HPLC to separate this super polar molecule from all of the shorter, untritylated and tritylated pieces. Here is an example of the HPLC chromatogram of an 11mer. Look at all the impurities!

RP-HPLC

Last, we use acid catalysis to cleave the trityl group and free our DNA for the next annealing step.

Annealing

With our project, we need dsDNA, called a duplex. Therefore, once the ssDNA is purified, we are ready to anneal the two complimentary single stranded pieces.

The annealing step involves mixing the strands in a 1:1 ratio, ramping up the heat to separate all the hairpins or unwanted DNA interactions, then slooooowly cooling to anneal the pieces together.