Resea ch G oup Gene ics
Me aba coding p o ocol: Pooling and Lib a y
P epa a ion o sequencing
This se up is o sequencing amplicons on Illumina pla o ms
NB always use in combina ion wi h he me aba coding lib a y p epa a ion. Checklis .
1) A e he PCR ge someone o pack you pla es in 2 plas ic bags, and pass i o you in he semi-
clean lib a y p epa a ion lab.
2) Th ow away he ou e bag and clean wi h bleach he inne bag a ound you samples.
3) Pu you samples in he idge (i no wo king s aigh away) o he wise place on he lab bench o
equilib a e o oom empe a u e while you clean he hoods and equipmen ollowing he p o ocol.
4) Take necessa y chemicals ou o he eeze 30 mins p io o wo k o de os . This akes app ox.
he same ime as UV’ing he hood a e cleaning, so do his simul aneously.
a. Ampu e beads
b. ETOH (in eeze )
c. Wa e ( idge)
d. S anda ds o qubi BR
Pooling and min elu e
1. Fo each pla e use a mul ichannel pipe e o pool 15 mic oli e s o each sample in o an eigh -
well s ip.
2. Combine he 8 well s ip in o an 1.5ML LOBIND eppendo ube, label his wi h he pla e
name, ma ke (e.g. COI, 12S), p e-pool, you name and he da e.
3. Fo each pool make 6 new eppendo ubes, each wi h 125mic oL o he pool and add
625mic oL o PB bu e (minelu e ki ).
4. Vo ex and b ie ly spin down he 6 ubes.
5. P epa e 3 minelu e spin columns, and add he 750mic oL sample PB bu e mix o hese
columns.
6. Spin he column a 13 RPM o 1 minu e
7. Th ow away he low h ough and hen add he emaining 3 ubes o sample PB mix o hese
3 minelu e columns.
8. Spin he column a 13 RPM o 1 minu e
9. Th ow away he low h ough and hen add 750 mic oL o PE bu e o he columns
10. Spin he column a 13 RPM o 1 minu e
11. Th ow away he low h ough
12. Spin he column a 13 RPM o 1 minu e
13. Add 15mic oL o EB bu e o each column making su e ha i only ouches he il e .
14. Spin he column a 13 RPM o 1 minu e
Au ho s:
Owen S. Wangens een
Ma a Tu on
Julie Bi z-Tho sen
Melissa B andne
Kim P æbel (kim.p aeb[email p o ec ed])
BFE/NFH - Resea ch G oup o Gene ics
Las upda ed: Janua y 2025
PO Box 6050 Langnes, N-9037 T omsø / 77 64 40 00 / pos mo a[email p o ec ed] / ui .no / o g. no. 970 422 528 2
15. Combine he 3 ubes wi h 15 mic oli e s in o one DNA lo-bind ube and label wi h he pla e
name, ma ke (e.g. COI, 12S), pos -pool, you name and he da e.
Quan i ica ion
1. Make a qubi B oad ange mas e mix by combining 1 mic oli e dye wi h 99 mic oli e bu e
o x = n+2 samples (n= numbe o samples+ numbe o s anda ds)
a. e.g. 10 samples n=12 x= 14
b. Dye = 14 mic o L. Bu e = 2786 mic oL
2. Add 198 mic oL o mas e mix (MM) o 10 sample ubes and 190 mic oL o MM o 2 s anda d
ubes.
3. Add 2 mic oL o each pla e pos pool o each qubi sample ube.
4. Add 10 mic oL o each s anda d o each qubi s anda d ube.
5. S a up he qubi luo ome e , selec dsDNA b oad ange and measu e he s anda ds i s hen
he samples.
Lib a y p epa a ion
This p o ocol is modi ied o he Qiaseq one s ep amplicon lib a y p epa a ion ki
Cleanup = Ampu e beads (BIOO me hod)
S a DNA concen a ion 3000 ng
1. P epa e ei he an 8 well s ip o pla e dependen on he numbe o lib a ies o p epa e.
2. Combine he ollowing in each well:
Componen
Volume/ eac ion (mic oL)
DNA (pos pool minelu e) 3000ng
X
wa e
31.5-X
4x 1-s ep Lib a y Bu e
12.5
Y adap e
4
1-s ep Amplicon enzyme mix
2
To al
50
3. Se pipe e o 20-30 mic oL mix he componen s by pipe ing up and down 10 imes
4. P og am a he mal cycle o incuba e a 25C o 1 hou .
Cleanup
Cleanup BIOO PCR ee 30 minu es
1. Measu e each sample using a 100mic oL pipe e se o 50 mic oL
2. Make sample up o 54.5 mic oL wi h H2O
3. Add 44 mic oL beads and pipe e up and down 10 imes o mix
PO Box 6050 Langnes, N-9037 T omsø / 77 64 40 00 / pos mo a[email p o ec ed] / ui .no / o g. no. 970 422 528 3
4. wai 5 minu es
5. Place he ubes on he magne ic ack o 2 minu es
6. Remo e and disca d supe na an
7. Wash 2 x wi h 80% ETOH ( eshly p epa ed) 200 mic oL
8. D y 3-5 mins
9. Resuspend wi h 57mic oL esuspension bu e
10. Wai 2 mins, place on s and and ans e supe na an o a clean ube 54.5 mic oL
11. Add 44 mic oL o beads
12. Incuba e 5 minu es hen place on magne
13. Remo e supe na an and disca d
14. Wash 2 x wi h 80% ETOH
15. D y 3-5 mins
16. Add 22.5 Mic oL esuspension bu e
17. Incuba e 2 minu es
18. Place on a magne o 5 minu es
19. T ans e 20 mic oL o lib a y
20.
