SureFood-PREP Animal

add 40 10 mg sample 400 l Lysis Buffer 40 l Proteinase K

add 550 l Wash Buffer I

Lysis

incubate at 52C, 30 min, continuous shaking

Wash DNA

centrifuge 12 000 rpm, 1 min, discard filtrate

centrifuge 12 000 rpm, 2 min

repeat Wash Step, discard filtrate

transfer the supernatant add 200 l Binding Buffer

Dry spin filter!

centrifuge 12 000 rpm, 2 min into new receiver tube

Adjust DNA binding conditions

vortex

transfer the spin filter into new receiver tube (clear) add 100 l pre-heated Elution Buffer into spin filter

Elute DNA
centrifuge 10 000 rpm, 2 min, discard spin filter

sample DNA

Bind DNA

transfer solution into spin filter in new receiver tube

centrifuge 12 000 rpm, 2 min, discard filtrate

SureFood-Animal ID: PCR-Pipetting scheme for n samples

PCR for detection of animal species

1 2 n

samples
add 2 l sample each: DNA No. 1 2 l sample DNA No. 2 2 l sample DNA No. n

(2n+4) x 18 l PCR Mix (Kit Code G) (2n+4) x 0,1 l Taq Polymerase

mix

add each 18 l of Taq PCR Mix add:

1c

2c

nc

inhibition controls

PCR

each 2 l positive-control DNA 2 (Kit Code D) positive control no-template control

controls for Taq PCR Mix

2 l positive control DNA 2 (Kit Code D) 2 l no-template control (Kit Code E)

add:

SureFood-Animal ID: Example plate layout for the detection of beef and porc in four different samples
Example for position of samples and controls in step 2.1 of the detection protocol. The samples and controls are further detected with the corresponding hybridization probes in step 2.4 . The colour development of all PCR and ELISA (hyp-) controls for correct sample interpretation is displayed in greyscale.

Binding of the PCR products and ELISA (hyb) controls in step 2.1

strip 1

strip 2

sample No. 1 sample No. 2 beef probe sample No. 3 porc probe

sample No. 4 inhibition control of sample No.1 inhibition control of sample No.2 inhibition control of sample No.3 inhibition control of sample No.4 PCR positivecontrol beef probe beef probe PCR -no-templatecontrol hyb-control (Kit Code F) hyb-control (Kit Code F) hybridization probe plus hybridization probe minus

Hybridization of probes in step 2.4