RGG DNA extraction protocol for water samples (eDNA)

Resea ch G oup Gene ics
Au ho s:
Kim P æbel (kim.p a[email p o ec ed])
Julie Bi z-Tho sen
No wegian College o Fishe y Science
Resea ch G oup o Gene ics, K. P æbel
Las upda ed: Sep embe 2023, edi KP
EXTRACTION PROTOCOL FOR EDNA FROM
STERIVEX FILTERS
The p o ocol is using he Qiagen Blood and Tissue ki
Guidelines and ad ises
• Follow he desc ip ions in he ‘Clean Lab Rou ines’ o how o en e he labs.
• Always s a a p ojec wi h a new ex ac ion ki . The ki can only be used o ha pa icula p ojec
and any emains a e b ough ou o he lab and o he B310 lab when he ex ac ions a e comple ed.
• Make su e ha he incuba o is se o 56oC be o e s a ing he ex ac ion wo k. The equipmen you
a e going o use o he ex ac ion p o ocol should always be cleaned.
• Make su e o check o any signs o c ys als in he ex ac ion bu e s when opening he ki in he
lamina low-hood. I any signs, place he bo le(s) in he incluba o a 56oC un il hey a e
comple ely dissol ed.
• Always shake Eppendo ubes ou o he bag, don’ pu you hand inside o i . Disca d any excess
ubes.
• Only open he bags con aining Eppendo ubes, o o he ubes, inside he lamina low-hood.
• Only use pipe e ips wi h ba ie s/ il e s and only open he boxes inside he lamina low-hood.
• Always ollow he wo k low o any p ecau ions gi en o he eDNA clean lab wo king ou ines.
• Always disca d ips/ ubes/glo es i you ha e he sligh es suspicion abou con amina ion (e.g. i
he ip ouches he able be o e en e ing a ube o bu e bo le).
• Always wo k wi h a leas one ex ac ion blanks pe ex ac ion ound (i.e. 24 samples). Howe e , i
you a e wo king wi h 22 samples o ex ac , hen o comple e he numbe o 24, you can wo k wi h
wo blanks.
• Always s a wi h he lowes concen a ion i.e. ai blanks and wa e blanks (i any) excep
ex ac ion blanks, which should be ea ed as any egula sample.
• I ex ac ing samples om se e al species/loca ions, s e ilize e e y hing be ween samples.
• Do no ouch he ends o he S e i ex il e s o he inside o he ube caps wi h hands o weeze s.
• Always be ca e ul when you open he Eppendo ubes no o ouch he inside o he cap. Hold
hem in you hand and lick hem open wi h he ip o you humb.
• MAKE SURE YOU HAVE ENOUGH TIPS ! - be o e en e ing he clean-lab. You will mainly use
1000µl ips bu also s ock up on 20µl and 200µl ones. You also need Eppendo ubes (bo h 1.5ml
and 2.0ml), 50ml alcon ubes. Always ha e enough o hese hings be o e you s a wo king.
DAY 0:
1. Follow he desc ip ions in he ‘Clean Lab Rou ines’ o how o en e he labs.
2. Since he dedica ed eDNA -80oC eeze is loca ed in a “con amina ed” a ea, he p e e ence is o ake
he samples ou he day be o e. Then, he day a e , you s a you ex ac ions by showing up in newly
washed clo hes and eshly showe ed.
3. Find clean glo es in he bags a he dedica ed eDNA eeze . The glo es do no need o be bleached since
he bags/ oom a e “di y”.
4. Find he desi ed bag o il e s o ex ac in he eDNA eeze .
Ex ac ion P o ocol – S e i ex il e s
2
5. When en e ing he ai lock in oom 358, swi ch glo es. Sp ay glo es wi h bleach. Sp ay he ou side o
bags libe ally wi h 10% bleach. Allow he sample bag o si w/ bleach o 5 minu es, hen d y hem.
6. Place he bags in one o he dedica ed plas ic boxes wi h lid in he idge in he ai -lock a 4oC o gen le
hawing. I akes app ox. 1-2 hou s.
7. Add he samples o he eDNA sample o e iew ile
DAY 1:
1. Follow he desc ip ions in he ‘Clean Lab Rou ines’ o how o en e he labs.
2. Clean he bags con aining he samples e y ho oughly using 10% bleach, wa e and hen 70 %E OH.
3. B ing hese cleaned bags in o he clean lab ollowing he clean lab ou ines.
4. To emo e excess wa e inside he il e s, place he inle o he il e (na ow end) in a 1.5 ml Eppendo
ube and gen ly slide il e and ube in o he 50 ml alcon ube ha con ained he il e (o in a new 50 ml
ube i samples we e s o ed in ziplock bags). I mo e han one il e is in he ube, label a new ube o
he second il e . When done wi h il e s om one species/s a ion, clean e e y hing again ( o ceps,
glo es, wo king su ace) wi h bleach, MilliQ wa e and e hanol, be o e p oceeding o he nex
species/s a ion.
5. Cen i uge he ubes a 1500 x g o 3 minu es o emo e he emaining seawa e om he il e s.
6. Make ex ac ion bu e solu ion o adding 2.5X he ecommended olume = 500µl pe il e .
o Recommended olume is 20µl P o einase K + 180µl Bu e ATL pe sample:
▪ 2.5 * 20µl P oK = 50µl
▪ 2.5 * 180µl ATL = 450 ul
▪ To al amoun o ex ac ion bu e pe sample = 500µl
▪ E.g. o 20 samples: 1000µl P oK, 9000µl ATL. Fi s , pipe e he 9ml wi h a s e ile glass pipe e
in o a clean 50ml alcon ube. Then pipe e 1 ml o P oK in o he same ube. Close wi h lid and
in e solu ion, a oiding oaming.
7. Add 500µl o he ex ac ion solu ion o each il e , s a ing wi h blanks, by pushing he 1000µl ip igh
in o he ou le end o he il e and gen ly aspi a ing he solu ion in o he il e . Take ca e ha all he
solu ion goes in o he il e . I he il e is clogged, hen aspi a e om he inle end o he il e .
8. Cap he il e s wi h s e ile caps. Make su e ha i s comple ely sealed.
9. MAKE SURE YOU LABEL ALL THE FILTERS CORRESPONDING TO THE TUBES, by w i ing
he label and he eplica e le e (A, B, C e c.) on he il e and co e wi h ape.
10. Place he il e s in o a o and as en hem wi h he elas ic band.
11. When done wi h all il e s, mo e he o a o o he incuba o o en (56C). Make su e ha he o a o is
mo ing a 6 pm and no hi ing he o en. Check he il e s a e a couple o hou s and lea e hem
o e nigh o he 2nd day o ex ac ions. Minimum 8-12 hou s incuba ion.
12. No e: Always use simila incuba ion ime o all il e s wi hin a p ojec . No e he ime o when
incuba ion in he incuba o o en s a ed.
DAY 2
13. En e lab and clean acco ding o he Clean Lab Rou ines.
14. Label all ubes needed o he p ocess: 2ml Eppendo ubes, spin columns and he inal 1.5ml Eppendo
ubes ha will hold he elu ed DNA (sample ID on op, and mo e de ails on he side including eplica ion
(A,B,C), dep h, da e o collec ion, da e o ex ac ion and you ini ials).
15. No e he ime when he il e s a e emo ed om he incuba o o en.
16. Reopen he sealed il e s and ans e hem o a ma ked 2ml ube inside a new 50ml alcon ube wi h he
inle acing down in o he 2ml Eppendo ube.
17. Cen i uge he 50ml ubes con aining he 2ml ubes and he il e s a 1700 x g o 3 minu es.
18. Remo e he il e om he 50ml ube and disca d i . Then ca e ully emo e he 2ml ube om he bo om
o 50ml ubes wi h a weeze holding he oo o he cap, wi hou ouching he cap i sel o he edge o
Ex ac ion P o ocol – S e i ex il e s
3
he ube opening. Close he 2ml ube and place i in a ack. Again, s a wi h he lowes concen a ion
(e.g. ai -> blank -> eal samples).
19. “Measu e” he app oxima e olume o 2-3 samples using a pipe e wi h NEW ips o each sample. Round
he mean olume o nea es 50µl.
20. Add an equal olume o he Bu e AL as he one de e mined abo e (7.) and ensu e o mix i wi h he
pipe e immedia ely using new ips o each sample.
21. Add an equal olume o 100% E OH as he one de e mined abo e (7.) and ensu e o mix i wi h he
pipe e immedia ely using new ips o each sample.
22. Vo ex and spin down he samples o make su e i is mixed and liquid om he cap is emo ed.
23. Place he spin columns in on o he samples in he ack.
24. T ans e 630µl o he sample in o co esponding spin column. Be ca e ul no o make any bubbles bu a
he same ime y no o lea e liquid in he ip because i is p ecious DNA.
25. Cen i uge he columns a 15.000 x g o 2 mins.
26. Disca d he collec ion ube wi h he low- h ough and ans e he spin column o a new collec ion ube.
Make su e he low- h ough has no spilled back o he column when you emo ed i om he cen i uge.
27. T ans e he es o he sample o he co esponding spin column. I mo e han 630µl, h ee ounds o
spinning a e equi ed.
28. Cen i uge he columns a 15.000 x g o 2 mins.
29. Disca d he collec ion ube wi h he low- h ough and ans e he spin column o a new collec ion ube.
Make su e he low- h ough has no spilled back o he column when you emo ed i om he cen i uge.
30. Add 500µl Bu e AW1 (check E OH has been added o bu e ) using new ips o each ube.
31. Cen i uge a 15000 x g o 2 mins.
32. Disca d he collec ion ube wi h he low- h ough and ans e he spin column o a new collec ion ube.
Make su e he low- h ough has no spilled back o he column when you emo ed i om he cen i uge.
33. Add 500µl Bu e AW2 and cen i uge o 4 mins a 20.000 x g.
34. While cen i uging, clean lowhood, pipe es, and pens wi h bleach, MilliQ and e hanol.
35. TAKE GREAT CARE ha no low- h ough is p esen on he sides o he spin columns. I so, spin he
columns again in a new collec ion ube a 20.000 x g o 2 mins. No e wha samples ha ha e been
cen i uged wice.
36. T ans e he spin-columns o he co esponding Eppendo ubes. Make su e ha he lid/ ap o he spin
column does no ouch he cap o he Eppendo ube o a oid con amina ion.
37. Add 75µl o Bu e AE o each spin columns. Make su e o add he bu e a he cen e o he memb ane
wi hou ouching he memb ane. Incuba e o 1 min, hen spin he samples a 20.000 x g o 2 mins.
38. Disca d he spin columns and ans e a 20µl aliquo o he ex ac ed DNA om each sample o a PCR
pla e o PCR s ips. I is e y impo an he pla e/s ip is labeled p ope ly wi h all necessa y in o ma ion
and wi h unique names (no jus ‘Pla e 1’!). I you a e using s ips, use an emp y pipe e ip box as ack.
W ap aliquo s in wo bags be o e empo a y s o age. Place he aliquo in he idge a 4C i you a e
ce ain i will be p ocessed wi hin he nex 2-3 weeks o in he aliquo eeze i longe .
39. S o e he es o he DNA as s ock in he eeze loca ed in he ex ac ion lab. S o e he 1.5ml ubes in a
c yobox ha you ha e pu chased a he s o e and b ough wi h you. Make su e o label he box p ope ly.
Pu he c yobox in wo bags be o e s o ing i and ONLY haw he s ock i absolu ely necessa y.
40. Clean he lamina low-hood and all equipmen acco ding o he clean-lab guidelines.