DNA Barcoding Workflow

1. DNA Barcoding Workflow Image used with permission from the Centre for Biodiversity Genomics (http://biodiversitygenomics.net)

2. To do DNA extractions you need: Skills: Vortexors Pipettors Microcentrifuge We’ll start with pipetting, after a reminder about units and conversions.

3. But first µL What is a µL? µL = microliter May also call it a “mic” (pronounced mike) 1000 µL 1 mL Is a microliter bigger or smaller than a drop of water? 1 mL x = 1000 µL 1 µL = 1 microliter = .001 mL = .000001 Liters 1 drop of water = ~50 µL How many µL are in a ml?

4. Pipetting For a good overview of basic pipetting, please show: https://www.youtube.com/watch?v=wcLfqKnAE-k Hyman et al. CURE-all: DNA barcoding in introductory biology

5. Pipetting Plunger • Pipettors measure small volumes (< 1 mL) accurately • Expensive – treat with care • Important points: • Parts of pipettor • Volume range • How do you adjust? • Always use with a pipette tip! • Each pipettor has unique pipet tips Tip ejector Volume gauge Shaft Pipette tip Hyman et al. CURE-all: DNA barcoding in introductory biology

6. Pipetting – Things NOT to do! • NEVER dial a pipettor too high or too low! • Volume range is listed on the side of pipettor • NEVER use a pipettor without a pipette tip. • NEVER turn a pipettor upside down with liquid in the tip! • NEVER leave a pipettor on its side. For a funny comic on pipetting please visit: http://www.biocomicals.com/ind_comicsV2.php?number=20120808 Remember, pipetting is a one person job – two hands on one body. Practice picking up and putting down tubes while you are pipetting!

7. Centrifuges must be balanced • Tubes must be evenly (symmetrically) arrayed around the center spindle Center spindle Three Tubes Four Tubes Four Tubes Two Tubes Balance tube Three Tubes https://www.youtube.com/watch?v=rwAoeNKNYR8

8. Centrifuge controls Speed controls • Adjust speed • Use arrows to adjust speed up and down • Max speed: 13.3 (or 13,300 x g) • Adjust time • Time displayed in minutes • For <1 min, set to 1 minute and then turn press stop button at desired time (or Quick spin) • Open lid • Start button • Will start centrifuge run • Stop button • Quick Spin (< 1 min) • Hold down – centrifuge will start, timer will count up • Release to stop centrifuge • You must use a lid • Prevents aerosols, noise • Lid will ‘Click’ into place Open Lid Quick Spin Adjust time Stop Start Centrifuge lid ‘Click’ into place!

9. Centrifugation tips • Arrange tubes in centrifuge with hinges on outside edge • Centrifugation today – two separate components • Supernatant = liquid on top • Pellet = stuff in bottom • Read your protocol to know which part to keep! • Sometimes you keep the pellet, sometimes the supernatant! Hinge (DNA is much smaller than this) Supernatant Pellet Hyman et al. CURE-all: DNA barcoding in introductory biology

10. Vortexors – Mix stuff up • Press tube onto blue portion • Vortex will vibrate and mix sample • Very common to: • Vortex tubes to mix, then • Centrifuge briefly (~5 s) to bring all contents to the bottom of tube Still need photographs of your specimen? Digital imager info next!

11. First Lab exercises – let’s get started • Work individually for all exercises • Pipetting Exercises First • Post-rinsing of small volumes – very important! • Always use the smallest pipettor for a volume • Pipettors are most accurate in their middle to upper range. • Perform the pipetting exercises – start at different places/exercises in the manual! • Centrifugation • Centrifuging silica resin • Vortexors • Avoiding contamination – no exercises, just common sense Remember your safety glasses!! Never leave pipette tip boxes open!!

12. DNA Extraction DNA is inside cells - must be extracted We recommend an image from Chromosomes, DNA and Genes: http://www.bbc.co.uk/education/guides/zp7thyc/revision/2 Hyman et al. CURE-all: DNA barcoding in introductory biology

13. Our “Crude” DNA Extraction • Separate cells by grinding. • Dissolve membranes, and some proteins with extraction buffer. • Precipitate DNA • Resuspend in a smaller volume. Precipitate, resuspend Your info here DNA Hyman et al. CURE-all: DNA barcoding in introductory biology other crap

14. Tips: DNA is everywhere, Contamination is Easy For a humorous DNA testing image, please visit: http://www.lemonharanguepie.com/2013/01/ https://www.youtube.com/watch?v=xxndtkXXBhE for a video on how barrier pipet tips work.

15. Preventing contamination • Wipe down your bench with cleaner • Wear gloves. • Change gloves if you suspect yours have been contaminated • Open and close all sample tubes and reaction plates carefully. • Keep test tubes and pipette tip boxes closed • Change pipette tips if tips will be touching things that will touch other samples • Clean scissors and other tools before and after using them Hyman et al. CURE-all: DNA barcoding in introductory biology

16. Hints • Clean bench, Goggles on, Gloves on • Add about ¼ - ½ pencil-eraser size of tissue • Avoid stomachs of large insects • Read the protocol carefully – use as a checklist. • Put used pestle in beaker on side bench. • Toss used vials in “used tips” container on bench • Don’t toss colored liquid vials • When all done, label your tube with colored tape and bring to me CM Inches Hyman et al. CURE-all: DNA barcoding in introductory biology