20.109(S13):DNA extraction (Day2)

Introduction

background on DNA extraction

background about sample choices -- bird species, time of year, cloacal vs stool maybe?

Protocols

Part 1: DNA extraction from bird stool, initiate

Note about increasing times for those with centrifuges that reach only 16K instead of 20K. And about 2 mL vs. 1.5 mL tube, not to mix up.

1. Obtain your [sign up previous time?] 100 μL bird stool sample from the teaching bench ice bucket.
   • One per person or per pair??
   • Work quickly to avoid thawing the frozen samples and keep on ice when not in use.
2. Immediately add 1.4 mL of buffer ASL and vortex for ~ 1 min, until the solution is homogeneous.
   • A few insoluble particulates may remain. Stop vortexing when the sample no longer visibly changes over a 20 sec interval.
3. Heat at either 70 °C for 5 min, using the heat block at the front bench.
4. Vortex for 15 sec and centrifuge for 1 min at 20,000 rcf.
5. Transfer 1.2 mL of supernatant into a fresh 2 mL tube.
6. Hold the foil-covered InhibitEX tablet over the tube, and gently push until the tablet pierces through the foil and falls into the tube.
7. Vortex until completely dissolved, which takes about 3 min for these samples.
8. Incubate 1 min longer (with no shaking), and then centrifuge for 3 min.
9. Transfer the supernatant (usually about X mL) to a 1.5 mL tube and again centrifuge 3 min.
10. In a fresh 1.5 mL tube, dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above and 200 μL buffer AL, pipetting to mix each time. Finally, add 10 μL chitinase.
11. Vortex for 15 sec (until solution is homogeneous) and incubate at 56 °C for about 2 hours [1.5??].

1. Quick-spin to recover sample that has condensed in the lid.
   • First time doing it, so explain quick-spinning with the usual text.
2. Add 200 μL of ethanol, mix, and transfer to a QIAamp spin column (atop its 2 mL collection tube).
3. Repeat centrifugation until all the sample has gone through the column.
4. Move spin column to a fresh 2 mL tube, add 500 μL buffer AW1, and spin 1 min.
5. Move spin column to a fresh 2 mL tube, add 500 μL buffer AW2, and spin 3 min.
6. Move to yet another fresh tube and spin 1 min to rid residual buffer.
7. Now transfer to a 1.5 mL tube and carefully pipet 200 μL buffer AE onto the membrane.
8. Incubate 1 min, then spin 1 min.

Part 2: Writing Across the Curriculum session

During the enzymatic incubation, you will have an introductory session with our writing faculty.

Part 3: DNA extraction from bird stool, complete

1. Quick-spin to recover sample that has condensed in the lid.
   • First time doing it, so explain quick-spinning with the usual text.
2. Add 200 μL of ethanol, mix by briefly vortexing, and transfer to a QIAamp spin column (atop its 2 mL collection tube).
3. Centrifuge for 1 min, and if necessary repeat the centrifugation until all the sample has gone through the column. (Unlikely in our case!)
4. Move the spin column to a fresh 2 mL tube, add 500 μL buffer AW1, and spin 1 min.
5. Move the spin column to a fresh 2 mL tube, add 500 μL buffer AW2, and spin 3 min.
6. In the meantime, trim and label epp
7. Move to yet another fresh tube and spin 1 min to rid residual buffer.
8. Now transfer the column to your trimmed, well-labeled 1.5 mL tube and carefully pipet 150 μL buffer of AE onto the membrane.
9. Incubate for 1 min, then spin for 1 min, cap, and store in your ice bucket. We will collect each sample and store it at -20 °C until next time.

For next time

Reagent list