Resea ch G oup Gene ics
Au ho s:
Owen S. Wangens een
Ma a Tu on
Julie Bi z-Tho sen
Kim P æbel (kim.p[email p o ec ed]o)
No wegian College o Fishe y Science
Resea ch G oup o Gene ics
Janua y 2020, 1.2 Ap 21KP
DNA AMPLIFICATION FOR METABARCODING
This se up is o sequencing on an Illumina pla o m wi h COI, 16s and 18s p ime s
using AmpliTag Gold Mas e Mix.
1) I you ha e ex ac ed you samples in he eDNA Ex ac ion lab, you b ing you aliquo s
wi h you in wo bags in o he PCR-se up lab. B ing also you sui om he ex ac 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 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.
5) The PCR-mix is as ollows:
REAGENT
VOLUME (µL)
AmpliTag Gold Mas e Mix
10.00
Bo ine se um albumin (BSA)
0.16
H2O
5.84
P ime s Mix (F + R)
2.0
DNA empla e
2.0
6) P epa e he Mas e Mix:
MASTERMIX
X1, µL
X8, µL
X100, µL
(96-WELL PLATE)
X?, µL
AmpliTag Gold
10
84
1050
BSA
0.16
1.34
16.8
H2O
5.84
49.06
613.2
(Use he space in he las column o do you calcula ion dependen on sample numbe ).
7) Gi e he Mas e Mix a ho ough o ex and spin i down.
8) Spin down he pla e wi h p ime s.
9) Add 15.9 µl o each well ha will be used in he PCR.
10) Add 2 µl o he p ime mix o each well.
11) Mo e o he o he hood be o e adding 2µl o he DNA empla e o each well wi h
Mas e Mix. (I you samples a e in he Gene ic lab B310, you add he 2µl o DNA in he
Me aba coding Wo ks a ion in B310).
12) Seal, w ap in bags, pu in idge while you clean he hoods ollowing he cleaning
p o ocol.
