BOSSLAB

Earlier today I emailed all of the participants in the recent Reading Genes workshop. Here is what I said:

Thanks so much for participating this weekend in the diy-genotyping workshop. I personally had a great time working with all of you and I learned a wonderful amount preparing and running the event. It was a bit of an experiment, and if some or all of the results are negative, rest assured that I will figure out what went wrong and invite you back for a successful second try.

Today I’m purifying everyone’s PCR products for sequencing. I’m using an enzyme I bought from Affymetrix (the ExoSAP-IT kit) to do the purification. The sequencing machine wants 12 uL (micro-liters) of each person’s purified PCR Product. So, I’ll take 12 uL of unpurified PCR mix from each person’s tube, add it to a new tube, and also add 4.5 uL of the magical ExoSAP-IT mix.

Theoretically, the included enzyme will chew up any remaining non-PCR-product DNA and remove their phosphate backbone. The genomic DNA we copied, the primers, and the DNA nucleotide building blocks that we built the copy from (the As, Gs, Cs, and Ts) will be changed from being nucleoTIDES to nucleoSIDES. I can’t give the sequencer tubes with active ExoSAP-IT, since it would probably mess up the sequencing reactions (we’re using “Sanger sequencing”, which is like a specialized kind of PCR + gel electrophoreses), so I’ll denature the ExoSAP-IT after it does its work by heating the entire mix to 80 C for 15 minutes.

So that’s the plan. I’ll let you know how the sequencing goes.