Tooth Extract Now Swollen

In this third meeting we started working, using a standard blood sample (without disease). To teach the technique, Adriana preparing a tube and we repeated it in triplicate.

Part One:
eppendorf tubes put in 500 ul of lysis solution. Add 300 ul of whole blood, taking care not to be formed bubbles. Propipeta mix for evacuation.
add 100 ul of 33% sucrose solution at the base of each tube without mixing the phases. We plugged the tubes and centrifuged at 13500 rpm for 2 minutes.
discarded the supernatant taking care not to remove the pellets.
We wash with 300 ul of PBS, mixing again for evacuation pipette.
add 100 ul of 11% sucrose solution at the base of each tube without mixing the phases. We plugged and re-centrifuge at 13500 rpm for 2 minutes. Again
discard the supernatant taking care not to remove the pellets.
add 75 ul of NaOH and vortex.
incubated in the thermocycler at 95 ° C for 14 minutes and 30 ° C for 1 minute. Centifugamos
briefly to lower condensation on the walls of the tube (use the "press").
neutralized with 10.5 ul of Tris / HCl and centrifuged 4 minutes at 14000 rpm.
DNA is found in the supernatant.
* Note: Each time a tip comes in contact with the sample should be excluded to avoid contaminating the solutions. Since we were using blood from one person, we put the necessary solution to each tube (without touching the sample) and after evacuation were mixing by pipette. When working with samples of different people we are going to use a tip for each sample.

Part Two: We
DNA to the third floor of the Academy, where was the spectrophotometer. We use a quartz cell plastic because the cells do not efficiently transmit ultraviolet light. We measured the absorbance at 230 nm, 260 nm (maximum absorption) and 280 nm.

At the conclusion of the pilot, Dr. Woods continued with the theoretical explanation about what they will do next week (PCR). Then he showed us the program Oligo, used to design primers that can identify potential problems and disadvantages of the design (the union of the chain, annealing temperature of melting, content G and C bases) for the best first time possible to make it.

today Photos: http://www.slide.com/r/IAbJpoLCwz-wsZcy8VSv8bYoiDEOg0_P?previous_view=mscd_embedded_url&view=original