446/546 handout 24 Aug. extracting nucleic acids AND homework.
TODAY, start experiment first to maximize available time, lecture will take place during
incubations
GENERAL ADVICE, READ INSTRUCTION BEFORE STARTING
Move carefully and deliberately, extractions are taking place in small volumes. It is easy to spill
the samples. Some of the solutions are designed to dissolve tissues and/or cells, so are potentially
harmful. Wear gloves, coat when handling chemicals. Gloves also protect the samples FROM
YOU, reducing the potential of contamination with your DNA/RNA, or introduction of enzymes
that degrade (foreign) nucleic acids.
ALWAYS spin eppendorf tubes with the hinge to the OUTSIDE, and you know that the pellet is
under the hinge.
INSTRUCTIONS:
(needed snail and parasite samples, pestle/tubes, CTAB, prot K, chloroform, isopropanol,
NH4OAc/EtOH, DNAzol, 100% EtOH, 75% EtOH, mQ water, waterbath at 60C, vortex,
centrifuges)
HAZARDOUS WASTE: GIVE TUBES WITH USED CTAB, DNAZOL TO INSTRUCTORS
DNA extraction from snail tissues
1) use SNAIL tissue sample (NOTE sample ID in YOUR LAB BOOK), transfer and place in
eppie tube.
2) use another regular eppie tube: ADD 10 microliter proteinase K (10 mg/ml stock) to 1000
microliter of CTAB
3) ADD 300 microliter CTAB/prot K to snail tissue
4) use plastic pestle to disrupt the snail tissue, leave no large tissue fragments.
5) ADD 300 microliter CTAB/prot K to the sample, mix by inversion
6) place (LABELED) sample in floatie in 60C waterbath for at least 30 minutes (mix every 10
minutes)
LECTURE
7) add 600 microliter chloroform (hood), mix
8) spin sample, BALANCE CENTRIFUGE, max rpm, rT, 5 minutes
9) take aqueous phase, UPPER layer (NOT LOWER, NOT INTERMEDIATE), transfer to clean
tube
10) add equal volume isopropanol to the aqueous phase in order to precipitate DNA, mix by gently
shaking, do you see DNA coils or flakes forming?
11) spin sample, BALANCE CENTRIFUGE, max rpm, rT, 5 minutes
12) remove isopropanol, add 1 ml Ammonium acetate/76% ETOH, incubate 15 minutes
IF YOU HAVE NOT ALREADY, START PARASITE DNA EXTRACTION
13) remove Ammonium acetate/76% ETOH, add 75% EtOH, mix (salt removal)
14) spin sample, BALANCE CENTRIFUGE, max rpm, rT, 5 minutes
15) remove ethanol, dry for 5 minutes by leaving tube open
16) dissolve pellet in 50 microliter milliQ water.
DNA extraction from parasite:
1) use parasite sample (NOTE sample ID in YOUR LAB BOOK)
2) Spin sample 1min max rpm, pipet of supernate.
2) ADD 1000 microliter DNAZOL (CONTAINS GITC, PHENOL, HAZARDOUS)
3) mix by vortexing, 3x 10 seconds
4) leave on bench top 2 minutes
5) add 500 microliter 100% EtOH to precipitate the DNA, mix by inversion, VIGOROUSLY for
15 seconds
6) leave 3 minutes rT
7) spin sample, BALANCE CENTRIFUGE, max rpm, rT, 5 minutes (MAY or may not see pellet)
8) remove supernate, DO NOT touch the bottom of the tube, leave some liquid in place. THIS IS
WHERE THE DNA IS!
9) add 500 microliter 75% EtOH, mix by inversion
10) spin sample, BALANCE CENTRIFUGE, max rpm, rT, 5 minutes, remove EtOH, DO NOT
touch the bottom of the tube, leave some liquid in place. THIS IS WHERE THE DNA IS!
12) repeat steps 9) and 10), dry for 5 minutes by leaving tube open, add 50 microliter milliQ water
to dissolve pellet.
STORE SAMPLES IN BOX in freezer.
Homework, answer in your note book
1) How does placing the disrupted snail tissues in CTAB/Prot K at 60 degrees C protect the DNA?
2) In the CTAB buffer, what is the final concentration of Proteinase K?
3) How does ethanol precipitation work?
a) the simple answer
b) and the physico-chemical answer, considering the function of salt