User s Guide. ExiPrep Dx Bacteria Genomic DNA Kit K-4414

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1 User s Guide ExiPrep Dx Bacteria Genomic DNA Kit K-4414

2 ExiPrep Dx Bacteria Genomic DNA kit Kit for the extraction of bacterial DNA from various clinical samples using ExiPrep 16 Dx User s Guide Version No.: 2.0 ( ) P l e a s e r e a d a l l t h e i n f o r m a t i o n i n b o o k l e t b e f o r e u s i n g t h e u n i t BIONEER CORPORATION 8-11 Munpyeongseo-ro, Daedeok-Gu, Daejeon, , Republic of Korea Tel: Fax: Homepage:

3 S a f e t y w a r n i n g s a n d p r e c a u t i o n s P l e a s e i n q u i r e B I O N E E R s C u s t o m e r S e r v i c e C e n t e r t o o b t a i n a c o p y o f t h e M a t e r i a l D a t a S h e e t ( M S D S ) f o r t h i s p r o d u c t. B e f o r e, d u r i n g a n d a f t e r u s e o f t h i s k i t a s d e s c r i b e d i n t h i s U s e r M a n u a l, a l l h a z a r d o u s m a t e r i a l s ( mi a. te e. r i a l s t h a t m a y h a v e c o m e i n c o n t a c t w i t h c l i n i c a l s a m p l e i n c l u d i n g t u b e s, t i p s a n d m a t e r i a l s s h o u l d b e p r o c e s s e d a n d d i s p o s e d o f a c c o r d i a p p l i c a b l e a n d a p p r o p r i a t e r e g u l a t i o n s o f t h e m u n i c i p a l i t y / g o v e r n m e n t i n w h i c h p r o d u c t i s b e i n g u s e d. P l e a s e r e a d t h e U s e r M a n u a l b e f o r e u s i n g t h i s K i t. P l e a s e c h e c k t h e i n t e g r i t y o f t i p s a n d o t h e r m a t e r isa ul psp l i e d w i t h t h i s k i t p r i o r t o u s e. A d h e r e t o g e n e r a l c l l a b o r a t o r y s a f e t y p r o c e d u r e s d u r i n g t h e e x p e r i m e n t. S o m e a p p l i c a t i o n s t h a t m a y b e p e r f o r m e d w i t h t h i s k i t m a y i n f r i n g e u p o n e x i s t i n g p a i n c e r t a i n c o u n t r i e s. T h e p u r c h a s e o f t hei s nk oi t id no c l u d e o r p r o v i d e a l i c e n s e t o p e r f o r m p a t e n t e d a p p l i c a t i o n s. U s e r s m a y b e r e q u i r e d t o o b t a i n a l i c e n s e d e p e n d i n c o u n t r y a n d a p p l i c a t i o n. W e d o n o t c o n d o n e n o r r e c o m m e n d t h e u n l i c e n s e d u s e o f p a t e n t e d a p p l i c a t i o n.

4 W a r r a n t y a n d l i a b i l i t y A l l B I O N E E R p r o d u c t s a r e m a n u f a c t u r e d a n d t e s t e d u n d e r s t r i c t q u a l i t y c o n t r o l p r o t B I O N E E R g u a r a n t e e s t h e q u a l i t y o f a l l d i r e c t l y m a n u f a c t u r e d p r o d u c t s d u r i n g t h e w a r p e r i o d o f o n e ( 1 ) y e a r f r o m d a t e o f p u r c h a s e. I f a ne y d i sscuo ev s e r ea d r r e l a t i n g t o c o m p r o m i s e i n p r o d u c t q u a l i t y, i m m e d i a t e l y c o n t a c t B I O N E E R s C u s t o m e r S e r v i c e C e n t (o r d e b i o n e e r. c o m). B I O N E E R d o e s n o t a s s u m e l i a b i l i t y f o r m i s u s e o f t h e p r o d u c t, i. e. u s a g e t off o r t h e p r a n y p u r p o s e s o t h e r t h a n i t s i n t e n d e d p u r p o s e a s d e s c r i b e d i n t h e a p p r o p r i a t e a p p l i c a b l e I n s t r u c t i o n s f o r. U B s I e O N E E R a s s u m e s l i a b i l i t y u n d e r t h e c o n d i t i o n t h a t t h e u d i s c l o s e s a l l i n f o r m a t i o n r e l a t e d t o t h e p r o b l e m t o B I O N E E R i n w ir ti ht it n e n 3 0 f o dr am y s o f o c c u r r e n c e. T r a d e m a r k s E x i P r e p D x a r e t r a d e m a r k s o f B I O N E E R C o r p o r a t i o n, R e p u b l i c o f K o r e a. C o p y r i g h t b y B i o n e e r C o r p o r a t i o n. A l l R i g h t s R e s e r v e d

5 C o n t e n t s 1. O v e r v i e w F e a t u r e s a n d p r i n c i p l e o f t h e t e. s. t C l i n i c a l s i g n i f i c a n c e C o n t e n t s o f t h e k i t S t o r a g e R e q u i r e d m a t e r i a l s B e f o r e y o u b e g i n A p p r o p r i a t e b e n c h u s e S a m p l e a l l o c a t i o n C l e a n i n g a n d m a i n t e n a n c e o f a c c e s s o r i e s W a r n i n g s a n d p r e c a u t i o n s S p e c i m e n h a n d l i n g S p e c i m e n c o l l e c t i o n S a m p l e s t o r a g e S a m p l e t r a n s p o r t I n t e r f e r i n g s u b s t a n c e s S a m p l e p r e t r e a t m e n t E x p e r i m e n t a l f l o w c h a r t N u c l e i c a c i d e x t r a c t i o. n E x p e r i m e n t a l s e t u p L o a d i n g c o n t r o l s a n d s a m p l e s R u n n i n g t h e e q u i p m e n t P r e p a r i n g f o r f o l l o-u w p w o r k i n g L i s t o f p r o t o c o l n u m b e r s T r o u b l e s h o o t i n g R e m a r k s R e f e r e n c e s E x p l a n a t i o n o f s y m b o l s

6 1. O v e r v i e w D e s i g n e d f o r u s e w i t h t E h x e i P r e p 1 6 D x, t h e E x i P r e p D x B a c t e r i a G e n o m i c D N A K i ts t h e t o t a l s o l u t i o n f o r t h e a c c u r a t e a n d r a p i d e x t r a c t i o n o f b a c t e r i a l D N A w i t h s a m p l e s (e. g. U r i n e, s w a b, b o d y f l ). u i d Ts h e s y s t e m i s d e s i g n e d f o r r a p i d e x t r a c t i o n o b a c t e r i a l D N A, d e l i v e r i n g u p t o 1 6 e x t r a c t e d s a m p l e s ( i n c l u d i n g t hc io n t r1 o. l5 s ) h o wu ir s f r o m r a w c l i n i c a l s a m p l e s. E x i P r e p D x B a c t e r i a G e n o m i c D N A K ic to n t a i n s a l l b u f f e r s a n d c o n s u m a b l e s n e c e s s a r y f o r e f f i c i e n t a n d e f f e c t i v e e x t r a c t i o n o f D N A f r o m c l i n i c a l s a m p l e s. T h e K i t i s i n t e n d e d f o r u s e b y a q u a l i f i e d c l i n i c a l d i a g n o s t i c s t a f f, t r a i n e d t o c o r r e c t l y a n a p p r o p r i a t e l y h a n d l e a n d m a n i p u l a t e c l i n i c a l s p e c i m e n s. A u s e r m u s t a l s o b e e q u i w i t h b a s i c e x p e r i m e n t a l t e c h n i q u e s r e q u i r e d f o r c o r r e c t e x e c u t i o n o f t h e e x p e r i d e s c r i b e d i n t h i s U s e r s G u i d e. 1

7 2. F e a t u r e s a n d p r i n c i p l e o f t h e t e s t E x i P r e p D x B a c t e r i a G e n o m i c D N A K i ts d e s i g n e d f o r t h e r aecxtti o n o f b a c t e r i a l g e n o m i c D N A f r o m v a r i o u s c l i n i c a l s a m p l e s. S o m e s a m p l e s m a y h a v e t o b e p r e t r e a t e d b e f e x t r a c t i o n. T h e p r e t r e a t e d s a m p l e s m u s t b e r e s u s p e n d e d w i t h t h e p r o v i d e d R e s u s p e n s i B u f f e r b e f o r e p r o c e e d i n g w i t h b a c t e r i a l g e n o m i c D N A oen x. t r a c t i B a c t e r i a l g e n o m idc N A i s e x t r a c t e d f r o m c l i n i c a l s a m p l e s b y lu ys si in s g ba u f f e r ( B u f f e r C a r t r i d g e 2 ) t o d i s r u p t t h e b a c t e r i a l s t Tr hu e c t eu xr pe o. s e d g e n o m i c D N A i s b o u n d t o t h e s u r f a c e o f s i l i c a m a g n e t i c b e a d s i n b i n d i ntg h e b uwf afse hr i. n g b u f f e r ( fbfue r C a r t r i d1 g e ) r i n s e s a n y i m p u r i t i e s t h a t m a y e x i s t, a n d t h e e l u t i o n b u f f e r e x t r a c t s t h e p u r e D N A b e a d s. [ D e s c r i p t i o n o f D N A e x t r a c t i o n u s i n g s i l i c a m a g n e t i c b e a d ] 2

8 ExiPrep TM Dx Bacteria Genomic DNA Kit 3. Clinical significance Bacteria are very common microbes living in the environment and in the human body. Bacteria are classified by many specifications, but can largely be divided into Gram-Positive and Negative, or aerobic and anaerobic bacteria. Most of them are not harmful but even beneficial to the humans. There still are great varieties of pathogenic bacteria depending on pathogenicity and properties. Disease-causing bacteria can be found in various parts of the human body including the oral cavity, gastrointestinal tract, genitourital tract, skin, etc. Pathologenic bacteria can cause tuberculosis, sexually transmitted diseases, food poisoning, etc., and countless other diseases are attributed to bacterial infection. Because of the sheer number of bacterial species, similar symptoms can arise from various source pathogens. Because antibiotic treatment depends on the type of bacteria, the accurate determination of infection source is critical. The evolution of nucleic acid amplification testing (NAT) methods have provided a new means of specifically detecting the cause of infection much more accurately and faster than the traditional culturing method. 4. Contents of the kit This Kit contains all reagents necessary for 96 reactions. 3

9 1 : K i t B o x L a b e l i n g & C o n t e n t C o u n t s ( T o t a l 9 6 t e s t s ) 1 e a 2 : B u f f e r C a r t r i d1 g e 6 e a 3 : B u f f e r C a r t r i d2 g e 6 e a 4 : U s e r s G u i d e 1 e a 5 : S a m p l e L o a d i n g T u b e s 9 6 e a E x i P r e T p M D x B a c t e r i a G e n o m i c D N A K i t 6 : S a m p l e L o a d i n g T u b e B o x 7 : D i s p o s a b l e F i l t e r T i p 8 : D i s p o s a b l e F i l t e r T i p B o x 9 : 0. 2 m l E l u t i o n t u b e 1 e a 9 6 e a 1 e a 1 2 e a 10 : E l u t i o n T u b e C a p 1 2 e a 11 : W a s t e T r a y 3 e a 12 : P r o t e c t i o n C o v e r 13 : C o n t a m i n a t i o n S h i e l d F i l t e r P a p e r s 14 : R e s u s p e n s i o n B u f f e r 1 2 e a 1 2 e a 1 e a 4

10 5. S t o r a g e E x i P r e T p M D x G e n o m i c D N A K i t s u t i l i z e o u r u n i q u e B u f f e r c a r t r i d g e s y s t e m. T h e B u f f e r c a r t r i d g e s c o n t a i n a l l c o m p o n e n t s f o r n u c l e i c a c i d e x t r a c t i o n, i n c l u d i n g : b i n d i n g b u f f e r, w a s h i n g b u f f e r, e l u t i o n b u f f e r a n d m a g n e t i c b e a d s o l u t i o n. E a c h B u f f e r c a r t r i d g e i s h e r m e tliy c asle a l e d w i t h a t-p h rl ey e s e a l i n g f o i l a n d t h e n w r a p p e d i n f i l m t o p r o t e c t a g a i n s t l e a k a g e, e v a p o r a t i o n a -c n d o nctr aoms is n a t i o n. T hbe u f f e r c a r t r i d g e s c a n b e s t o r e d d r y a t r o o m t e m p e r a t u r-3 e 5 ( 1 ) 5 f o r u p t o 1 y e a r s f r o m t h e d a t e o f d e l i v e r y, perd o vtihde y r e m a i n s e a l e d. R e s u s p e n s i o n b u f f e r m a y f o r m p r e c i p i t a t e s d u r i n g s t o r a g e. I f t h i s o c c u r s, p l e a s e w a r m t h e R e s u s p e n s i o n b u f f e r t o u n t i l t h e p r e c i p i t a t e s a r e c o m p l e t e l y d i s s o l v e d. 6. R e q u i r e d m a t e r i a l D i s p o s a b l e p o w d e r-f r e e g l o v e s A p p r o p r i a t e v o l u m e p i p e t t e s S t e r i l i z e d, f i l t e r e d p i p e t t e t i p s 1. 5 m l m i c r o c e n t r i f u g e t u b e s V o r t e x m i x e r D e s k t o p c e n t r i f u g e E x i P r e p 1 6 D x ( C a t. N o -5. : 0 5A0, B i o n e e r C o r p. ) 5

11 7. B e f o r e y o u b e g i n W e r e c o m m e n d t h a t s e v e r a l p r e c a u t i o n a r y m e a s u r e s t a kr e n t o e n s u r e p r o p e r r e s u l t s a r e o b t a i n e d A p p r o p r i a t e b e n c h u s e P r e s s u r i z e d b e n c h e s a r e d i v i d e d pion st io t i v e p r e s s u ra en d n e g a t i v e p r e s s u r e b e n c h e s. P o s i t i v e p r e s s u br e n c h e s (e. g.c l e a n b e n c h e s e t c. ) a r e w o r k s p a c e s w h e r e f i l t e r e d a i r f l o w s o u t w a r d, t h u s k e e p i n g a c l e a n e n v i r o n m e n t w i t h i n t h e w o r k s N e g a t i v e p r e s s u r eb e n c h e s ( e. g. B i o s a f e t y C a b i n e t s, f u m e h o o d s e t c. ) s e n d a i r f t h e l a b o r a t o r y s p a c e o u t s i d e. I n o t h e r wr o rfd ls o, w s a ii n w a r d. T h i s a i r f l o w p r e v e n t s d a n g e r o u s s u b s t a n c e s f r o m c o n t a m i n a t i n g t h e l a b o r a t o r y e n v i r o n m e n t. W h e n o p e n i n g s t e r i l i z e d c o n t a i n e r s s u c h a s B u f f e r C a r t r i d g e s i n c l u d e d w i t h i n t t h e w o r k s h o u l d b e c o n d u c t e d i n a p o s i t i v e p r e s s u r e e n v i tr o n m pe rn et v e n t e n v i r o n m e n t a l c o n t a m i n a n t s f r o m e n t e r i n g a n d f o u l i n g t h e s t e r i l e s u p p l i e s. b e f o r e r e m o v i n g s u p p l i e s f r o m t h e p o s i t i v e p r e s s u r e e n v i r o n m e n t, p l e a s e t a p p r o p r i a t e m e a s u r e s t o a s s u r e t h a t s t e r i l i t y o f t h e m a t e r i a l s a r e p r e s e r v e i n t ro d u c e d i n t o a s t a-p t ir ce s s u r e e n v i r o n m e n t. O n t h e o t h e r h a n d, w h e n h a n d l i n g c l i n i c a l s a m p l e s ( e s p e c i a l l y o f h i g h p a t h o g e n i i t i s c r i t i c a l f o r t h e s a f e t y o f t h e o p e r a t o r a n d o t h e r s t a f f m e m b e r s t h a t a l l ( i. e. d e c a p p i n g, p i p e t t i n g, g c aop f p i nc l i n i c a l s a m p l e s a n d c o n t a i n e r s ) b e c o n d u c t e d w i t h i n a n e g a t i v e p r e s s u r e b e n c h a n d p r e f e r a b l y, a f i l t e r e d b e n c h r a t e d f o c l a s s i f i c a t i o n o f p a t h o g e n s t h a t a r e b e i n g h a n d l e d, s u c h a -r s aa t e pd r obpieo rsla yf e t y c a b i n e t S a m p l e a l l o c a t i o n A l t h ou g h t h e p r o d u c t c o n t a i n s a l l n e c e s s a r y c o n s u m a b l e s f o r a s u c c e s s f u l e x p e r i m t h e n u m b e r o f c o n s u m a b l e s s u p p l i e d w i t h i n a s s u m e t h a t a t l e a s t t w o s t r i p s w o r t h w e l l s ) b e r u n a t a t i m e. W e r e c o m m e n d a t t h e v e r y l e a s t t h a t s a m p l e s b e r u-w n e lbl y s8t r i p u n i t s t o p r e v e n t t h e u n f o r t u n a t e c i r c u m s t a n c e o f c o n s u m a b l e s r u n n i n g o u t rb e af go er ne t s. I f t h e s i t u a t i o n d i c t a t e s t h a t o n e o r t w o s a m p l e s b e er mu en r g( ee n. cg y. s a m p l e s ), p l e a s e c o n t ac t y o u r l o c a l B I O N E E R a g e n t t o o r d e r a d d i t i o n a l c o n s u m a b l e s. 6

12 7. 3. C l e a n i n g a n d m a i n t e n a n c e o f a c c e s s o r i e s T h e g r e a t e s t s o u r c e o f c o n t a m i n a t i o n i s -ih nu dm ua cn e d. T o p r e v e n t u n i n t e n d e d c o n t a m i n a t i o n o f r e a g e n t s, w e r e c o m m e n d t h a t t h e f o l l o w i n g g u i d e l i n e s b i m p l e m e n t e d i n c l e a n i n g a n d m a i n t a i n i n g t h e v a r i o u s a c c e s s o r i e s i n v o l v e d w i t h k i : D i s c a r d a l l l i q u i d s, p l a s t i c c o n s u m a b l e s, u s e d w i p e r s e t c. i n a p p r o p r i a t e c o n t a i n e r s a c c o r d i n g a l l a p p l i c a b l e t o l o c a l a n d n a t i o n a l r e g u l a t i o n s W. a s t e t r a y 1 D i s c a r d t h e l i q u i d w a s t e i n a n a p p r o p r i a t e c o n t a i n e r. 2 R i n s e w i t h d i s t i l l e d w a t e r a n d d i s c a r d t h e w a s t e i n a n a p p r o p r i a t e c o n t a i H. o l e P u n c h e r ( 6 H o l e P u n c h o r M u l t i P u n c h ) T h e h o l e p u n c h e r m a y b e a s o u r c e o f c o n t a m i n a t i or n r ae fp te ea t e d u s e. K e e p t h e h o l e p u n c h e r w i t h i n a c l e a n b e n c h a t a l l t i m e s t o p r e v e n t c o n t a m i n a t i o n. A d t o t h e f o l l o w i n g c l e a n i n g g u i d e l i n e s t o p r e v e n t p o t e n t i a l c o n t a m i n a t i o n o c c u r r i n g. C l e a n t h e p u n c h i n g e d g e s w i t h c l e a n i n g l i q u i d s c a p a b l e doif n g d engu rc al e i c a c i d s e (. g.5 % n i t r i c a c i d e t c. ) : T h e p u n c h i n g e d g e s a r e e x t r e m e l y s h a r p a n d m a y c a u s e w o u n d i n g i f h a n d l e d w i t h o u t c a u t i o n. 1 R i n s e w i t h d i s t i l l e d w a t e r. 2 A i r d r y c o m p l e t e l y i n a p o s i t i v e p r e s s u r e b e n c h T. i p p r o t e c t o r 1 W e t a l i -f n tr e e c l o t h o r t i s s u e w i p e r w i t h n u c l e i c a c i d c l e a n d i n cg l el ai n q u i d t h e t i p p r o t e c t o r. 2 R i n s e t h e i n s i d e w i t h -f a rleie n tc l o t h o r w i p e r w e t w i t h d i s t i l l e d w a t e r. 3 A i r d r y c o m p l e t e l y i n a p o s i t i v e p r e s s u r e b e n c h. 7

13 C. o n t a m i n a t i o n s h i e l d D i s c a r d t h e f i l t e r p a p e r o n t h e C o n t a m i n a t i o n S h i e l d i n a n a p p r o p r i a t e c o n t a d e e m e d n e c e s s a r y. W e t a l i -f n tr e e c l o t h o r t i s s u e w i p e r w i t h c l e a n i n g e. l g i. eqt uh ia d n o ( l, s o a p e t c. ) a n d c l e a n t h e C o n t a m i n a t i o n S h i e l d. 1 R i n s e w i t h a l-f i rn et e c lt oh o r w i p e r w e t w i t h d i s t i l l e d w a t e r. 2 A i r d r y c o m p l e t e l y i n a p o s i t i v e p r e s s u r e b e n c h. 3 R e p l a c e t h e f i l t e r p a p e r b e f-ao tr te a cr he i n g t o t h e i n s t r u m e n t E. l u t i o n t u b e r a c k 1 W a s h w i t h c l e a n i n g l i q u i e d. g.e( t h a n o l, s o a p e t c. ). U s e a -t c oi tp t oa np p l i c a t o r t o c l e a n t h e i n s i d e o f t h e w e l l s i f n e c e s s a r y. 2 R i n s e w i t h d i s t i l l e d w a t e r. 3 T a p t h e r a c k u p s i-dd oe w n o n a n a b s o r p t i v e s u r f a c e t o e x p e l r e s i d u a l w a t e r f r o m t h e w e l l s. 4 A i r d r y c o m p l e t e l y i n a p o s i t i v e p br e ns cs h u. r e D. i s p o s a b l e t i p r a c k 1 W a s h w i t h n u c l e i c a c i d c l e a n i n g s o l u t i o n. U s e -ta i p c o at pt po ln i c a t o r t o c l e a n t h e i n s i d e o f t h e w e l l s i f n e c e s s a r y. 2 R i n s e w i t h d i s t i l l e d w a t e r. 3 T a p t h e r a c k u p s i-dd oe w n o n a n a b s o r p t i v e s u r f a c e t o e x p e l r e s i d u a l w a t e r f r o m t h e w e l l s. 4 A i r d r y c o m p l e t e l y i n a p o s i t i v e p r e s s u r e b e n c h S. a m p l e t u b e r a c k 1 D i s p o s e t h e s a m p l e t u b e i n a n a p p r o p r i a t e w a s t e c o n t a i n e r. 2 W a s h w i t h c l e a n i n g l i q u i e d. g.e( t h a n o l, s o a p e t c. ). U s e a -t c oi tp t oa np p l i c a t o r t o c l e a n t h e i n s i d e o f w e tl hl es i f n e c e s s a r y. 3 R i n s e w i t h d i s t i l l e d w a t e r. 4 T a p t h e r a c k u p s i-dd oe w n o n a n a b s o r p t i v e s u r f a c e t o e x p e l r e s i d u a l w a t e r f r o m t h e w e l l s. 5 A i r d r y c o m p l e t e l y i n a p o s i t i v e p r e s s u r e b e n c h. 8

14 8. W a r n i n g s a n d p r e c a u t i o n s B u f f e r C a r t r i d1 g e Risk Hazard Clssification : Volatile liquid : Class 2 Extreme eye damage/eye irritation : Class 2 Carcinogen : Class 1A Reproductive Cell Mutation : Class 1B Reproductive toxicity : Class 1A Specific target organ toxicity (single exposure) : Class 3 (Respiratory systemirritation) Specific target organ toxicity (single exposure) : Class 3 (Anesthetic) Specific target organ toxicity (repeat exposure) : Class 1 Risk Hazard phrase : H225, H319, H335, H336, H340, H350, H360, H372 Prevention : P201,P202, P210, P233, P240, P241, P242, P243, P260, P261, P264, P270, P271, P280, P281 Response : P303+P361+P353, P304+P340, P305+P351+P338, P308+P313, P312, P314, P337+P313, P370+P378 Storage : P403+P233, P403+P233, P403+P235, P405 / Disposal : P B u f f e r C a r t r i d2 g e Risk Hazard Clssification : Volatile liquid : Class 2 Acute toxicity (oral) : Class 4 Skin corrosion/irritation : Class2 Extreme eye damage/eye irritation : Class 2 Carcinogen : Class 1A Reproductive Cell Mutation : Class 1A Reproductive toxicity : Class 1A Specific target organ toxicity (single exposure) : Class 3 (Respiratory systemirritation) Specific target organ toxicity (single exposure) : Class 3 (Anesthetic) Specific target organ toxicity (repeat exposure) : Class 1 Risk Hazard phrase : H225, H302, H315, H319, H335, H336, H340, H350, H360, H372 Prevention : P201,P202, P210, P233, P240, P241, P242, P243, P260, P261, P264, P270, P271, P280, P281 Response : P301+P312, P302+P352, P303+P361+P353, P304+P340, P305+P351+P338, P308+P313, P312, P314, P321, P330, P , P337+P313, P370+P378 Storage : P403+P233, P403+P235, P405 / Disposal : P501 9

15 9. S p e c i m e n h a n d l i n g A l l s a m p l e s m u s t b e t r e a t e d a s p o t e n t i a l b i o h a z a r d s S p e c i m e n c o l l e c t i o n E x i P r e p T M D x B a c t e r i a G e n o m i c D N AK i t i s o p t i m i z e d f o r b a c t e r i a l D N A e x t r a c t e d f r o m v a r i o u s c l i n i c a l s a m p l e s. B a c t e r i a l D N A s h o u l d b e i s o l a t e d f r o m c l i n i c a l s a m p l o n e d a y a f t e r c o l l e c t i o n. : O n l y u s e p r e s e r v a t -f i vree e c o n t a i n e r s S a m p l e s t o r a g e T h e c l i n i c a l s a m p l e s c a n b e s t o r e d u p t o a d a y a t 2 ~ 8 C. F o r l o n g p e r i o d o f t h e c l i n i c a l s a m p l e s s h o u l d b e s t o-2 r 0 e d ~ -8 a t 0 C i n a l i q u o t s S a m p l e t r a n s p o r t A l l s a m p l e s s h o u l d b e t r a n s p o r t e d i n a s h a t t e r p r o o f t r a n s p o r t c o n t a i n e r t o p p o t e n t i a l i n f e c t i o n f r o m s a m p l e l e a k a g e. S a m p l e s s h o u l d b e t r a n s p o r t e d a c c o r d i n l o c a l o r n a t i o n a l g u i d e l i n e s r e g a r d i n g b i o h a z a r d t r a n s p o r t a t i o n I n t e r f e r i n g s u b s t a n c e s T h e c l i n i c a l s a m p l e s m a y c o n t a i n s e v e r a l P C R i n h i b i t o r s. D u r i n g be ax ct tr ea rc it ai l o n o f D N A, P C R i n h i b i t o r s s h o u l d b e r e m o v e d t o g e t r e l i a b l e d e t e c t i o n o f D N A S a m p l e p r e t r e a t m e n t S p u t u m S a m p l e a m o u n t : > 1 ml S t o r a g e c o n d i t i o n s a n d p e r i o d : R e f r i g e r a t e d ( 7 d a y s ), F r o z e n ( > 7 d a y s ) M a t e r i a l s : T a b l e t o p c e n t r i f u g e ( u p t o 1 3, r p m ), v o r t e x m i x e r, 1 0 N N a O H, 1 ( p H 6. 8 ~ 7. 2 ) M e t h o d 1 P o u r 1 / 1 0 v / v 1 0 N N a O H i n t h e s p u t u m s a m p l e c o n t a i n e r a n d v o r t e x h a r d f o r m i n u t e s. 2 I n c u b a t e a t r o o m t e m p e r a t u r e ( 2 5 C ) f o r 1 5 m i n u t e s. 10

16 : I f t h e s a m p l e i s e x c e s s i v e l y v i s c o u s, l o a d u p t o t w i c e t h e v o ol r u m e o f N a O H e x t e n d t h e i n c u b a t i o n p e r i o d u p t o 3 0 m i n u t e s. I n c a s e o f t h a t t h e s a m p l e i s v i s c o u s i n s p i t e o f d o u b l e d v o l u m e o f N a O H, t r a n s f e r t h e s a m p l e t o a c o n i c a l a n d a d d e q u i v a l e n t a m o u n t o f P B S o r D E P C w a t e r t o t h e s a m p l e t h e n v o r t e x t h e t u b e. 3 V o r te x t h e s a m p l e h a r d f o r 1 m i n u t e. T r a n sml f e r t 1 o. 5 a s t e r i l i z e d ml 1. 5 m i c r o c e n t r i f u g e t u b e. 4 C e n t r i f u g e a t 9, r p m f o r 5 m i n u t e s a n d c o m p l e t e l y r e m o v e t h e s u p e r n a t a n : I f a w h i t e l a y e r f o r m s a b o v e t h e c e l l p e l l e t, r e m o v e t h e l a y e r w i t h t h e s u p e r n a t a n t a n d l e a v e t h e c e l l p e l l e t. 5 A d d 1 ml o f 1 x P B S i n t o ml 1. 5 m i c r o c e n t r i f u g e t u b e a n d v o r t e x f o r 3 0 s e c o n d s t o w a s h t h e c e l l p e l l e t. C e n t r i f u g e a t 9, r p m f o r 5 m i n u t e s a n d c o m p l e t e l y r e m o v e t h e s u p e r n a t a n t. R e p e a t t h i s w a s h i n g s t e p 2 ~ 3 t i m e s. : I f a w h i tae y elr f o r m s a b o v e t h e c e l l p e l l e t, r e m o v e t h e l a y e r w i t h t h e s u p e r n a t a n t a n d l e a v e t h e c e l l p e l l e t. 6 T r a n s f e r 4 0 μl 0 o f R e s u s p e n s i o n B u f f e r, 1 x P B S o r n o r m a l s a l i n e a n d c o m p l e t e l y d i s s o c i a t e t h e c e l l p e l l e t u s i n g a p i p e t t e. 7 B r i e f l y s p-d i on w n a t 6, r p m f 5 o r s e c o n d s. T h e s a m p l e i s n o w r e a d y t o b e u s e d B o d y f l u i d s S a m p l e t y p e : P a r t i-f c lr e e b o d y f l u i d s, e. g. B A L, P l e u r a l f l u i d, S a l i v a e t c. S a m p l e a m o u n t : > 5 ml S t o r a g e c o n d i t i o n s a n d p e r i o d : R e f r i g e r a t e d ( 7 d a y s ), F r o z e n ( > 7 d a y s ) M a t e r i a l s : T a b l e t o p c e n t r i f u g e ( u p t o 1 3, r p m ), v o r t e x m i x e r, 1 0 N N a O H, 1 ( p H 6. 8 ~ 7. 2 ) M e t h o d 1 V o r t e x t h e s a m p l e c o n t a i n e r h a r d a n d t r a n s f eml r 5 ~ o 1f 0 s a m p l e t o a c o n i c a l t u b e. : I f t h e s a m p l e i s v i s c o u s, t r a n s f e r μl 1 0 N N a O H a n d i n c u b a t e a t r o o m t e m p e r a t u r e f o r 1 5 m i n u t e s. I f t h e i n c u b a t e d f l u i d i n c l u d e s f l o a t i n g p a r e m o v e t h e p a r t i c l e s b e f o r e u s e. 2 C e n t r i f u g e a t 3, r p m f o r 2 0 m i n u t e s a n d c o m p l e t e l y r e m o v e t h e s u p e r n a t a n t. 3 A d d 1 ml o f 1 x P B S a n d v o r t e x f o r 3 0 s e c o n d s a n d t r a n s f e r t o a s t e r i l i z ml m i c r o c e n t r i f u g e t u b e. C e n t r i f u g e t h e s a m p l e a t 9, r p m f o r 5 m i n u t e s 11

17 c o m p l e t e l y r e m o v e t h e s u p e r n a t a n t. : I f a w h i t e l a y e r f o r m s a b o v e t h e c e l l p e l l e t, r e m o v e t h e h e l a y e r w i s u p e r n a t a n t a n d l e a v e t h e c e l l p e l l e t. 4 T r a n s f e r μl o f R e s u s p e n s i o n B u f f e r, 1 x P B S o r n o r m a l s a l i n e a n d c o m p l e t e l y d i s s o c i a t e t h e c e l l p e l l e t u s i n g a p i p e t t e. 5 B r i e f l y s p-d i on w n a t 6, r p m f o r 5 s e c o n d s. T h e s a m p l e i s n o w r e a d y t o b u s e d U r i n e ( S a m p l e L o a d i n g T u b e ) S a m p l e A m o u n t : > 1 0 ml ( f i r s t m o r n i n g u r i n e i s r e c o m m e n d e d ) S t o r a g e c o n d i t i o n s a n d p e r i o d : R e f r i g e r a t e d ( 7 d a y s ), F r o z e n ( > 7 d a y s ) M a t e r i a l s : S w i n g r o t o r c e n t r i f u g e ( u p t o 3, r p m ), v o r t e x m i x e r M e t h o d 1 C e n t r i f u g e t h se a m p l e t u b e a t 3, r p m f o r 2 0 m i n u t e s a n d r e m o v e t h e s u p e r n a t a n t. 2 T r a n s f e r μl o f R e s u s p e n s i o n B u f f e r, 1 x P B S o r n o r m a l s a l i n e a n d c o m p l e t e l y d i s s o c i a t e t h e c e l l p e l l e t u s i n g a p i p e t t e. : I f e x c e s s s e d i m e n t c a u s e s v i s c o s i t y i s s u e s, b u f f e r v o l u m e c a n b e i n c r e a s e d u p t o t h r e e t i m e s m o r e t h a n t h e s e d i m e n t v o l u m e. 12

18 ( S a m p l e L o a d i n g T u b e ) S w a b S a m p l e u n i t : 1 s w a b S a m p l e c o l l e c t i o n m e t h o d T a k e a s t e r i l i z e d b r u s h f o r t h e u s e o f s p e c i f i c s a m p l e c o l l e c t i o n, a n d i n s e r t i n c h ) i n t o tuhre e t h r a ( u s e u r o l o g i c a l -o nu ls y e b r u s h ), V a g i n a l / C e r v i c a l, t h r o a t ( u s e r e s p i r a t o r y u-o s en l y b r u s h ) e t c. S m o o t h l y t w i s t t o c o a t t h e b r u s h, a n d k e e p t h e b r u s h i n i t s c o n t a i n e r. S t o r a g e c o n d i t i o n s a n d p e r i o d : R e f r i g e r a t e d ( 7 d a y s ), F r o z e n ( > 7 d a y s ) M a t e r i a l s : T a b l e t o p c e n t r i f u g e ( u p t o 1 3, r p m ), v o r t e x m i x e r, 1 X P B S ( p H 6. 8 ~ 7. 2 ) M e t h o d 1 V o r t e x t h e s a m p l e c o n t a i n e r h a r d f o r 3 m i n u t e s. T r a μl n s f eor f 5f0l0 u i d i n t o a s t e r i l i z e d ml 1. 5 m i c r o c e n t r i f u g e t u b e. : I f t h e s a m p l e i s d r i e d i n t h e a b sb eu nf cf e r o, f t h e s a m p l e c a n n o t b e u s e d f o r a d i a g n o s t i c p u r p o s e. : I f v i s c o u s s a m p l e s c a u s e t i p s t o c l o ml g, ap db d S 2 b u f f e r a n d v o r t e x t h e s a m p l e b e f o r e u s e. 2 C e n t r i f u g e t h e s a m p l e a t 1 3, r p m f o r 5 m i n u t e s a n d c o m p l e t e l y r e m o v e t h e s u p e r n a t a n t. 3 T r a n s f e r μl o f R e s u s p e n s i o n B u f f e r, 1 x P B S o r n o r m a l s a l i n e a n d c o m p l e t e l y d i s s o c i a t e t h e c e l l p e l l e t u s i n g a p i p e t t e. : H a r d p a r t i c l e s t h a t c a n n o t b e r e s u s p e n d e d m a y c a u s e t i p s t o c l o g d u r i n g t n u c l e i c a c i d e x t r a c t i o n p r o c e s s. O n l y u s e c o m p l e t e l y r e s u sl pu et in do ne. sd s o 13

19 S t o o l S a m p l e a m o u n t : 5 0 mg ~ mg / μl S a m p l e c o l l e c t i o n m e t h o d ( S a m p l e L o a d i n g T u b e ) T a k e a 1 ~ 2 g / ml 2 p o r t i o n o f s t o o l w h e r e p u s, b l o o d o r m u c u s i s p r e s e n t a n d t r a n s f e r i n t o a d r y, c l e a n c o n t a i n e r. S t o r a g e c o n d i t i o n s a n d p e r ir oe d f : r i g e r a t e d ( 3 d a y s ) M a t e r i a l s : T a b l e t o p c e n t r i f u g e ( u p t o 1 3, r p m ), v o r t e x m i x e r, s h a k i n g i n c u b a t o r ( 6 0 ), S t o o l l y s i s b u f f e r ( s o l d s e p a r a t e l y ), P r o t e i n a s e K ( s o l d s e p a M e t h o d 1 T r a n s f e r 5 0 ~ mg s a m p l e i n t o a s t e r i l i z eml d 1. m 5 i c r o c e n t r i f u g et u b e a n d a d d μl S t o o l l y s i s b u f f e r w i μl t h 1P0 r o t e i n a s e K ( mg 4 0 / ml ). 2 V o r t e x t h e t u b e h a r d f o r 1 m i n u t e. T h e n p l a c e t h e t u b e sih n a ka i n6g 0 i n c u b a t o r u n t i l t h e s a m p l e i s c o m p l e t e l y d i s s o c i a t e d ( a p p r o x. 3 0 m i n u t e s ) 3 A f t e r c o m p l e t e l y s i s, c e n t r i f u g, e 0 0a0 t r1p2m f o r 5 m i n u t e s. 4 T h e s u p e r n a t a n t m a y b e u s e d f o r e n s u i n g e x p e r i m e n t s. 14

20 1 0. E x p e r i m e n t a l f l o w c h a r t 15

21 1 1. N u c l e i c a c i d e x t r a c t i o n F o r e x t r a c t i o n o f n u c l e i c a c i d s, p l e a s e f o l l o w t h e i n s t r u c t i o n s b e l o w o n h o w t o E x i P r e p D x B a c t e r i a G e n o m i c D N A K i t a n d te hx e i P r e p 1 6 D x. T h e e x t r a c t i o n p r o c e s s i n c l u d e s s a m p l e p r e p a r a t i o n, e x t r a c t i o n p r o c e s s s e t u p, p i p e t t i n g s a m p l e s a n d e x e c t h e e x t r a c t i o n b y o p e r a t i n g Etxhi e P r e p 1 6 D x. T h e e x t r a c t e d b a c t e r i a l D N A i s a u t o m a t i c a l l y ld oe ad i n t o t h e E l u t i o n t u b e s E x p e r i m e n t a l s e t u p 1 T u r n o n t h e E x i P r e p 1 6 D x b y p r e s s i n g t h e m a i n p o w e r b u t t o n l o c a t e d a t t h e f r o n t o f t h e i n s t r u m e n t. P r e s s t h e S T A R T I N G i m a g e d i s p l a y e d o n t h e L C i n i t i a t e i n s t r u m e n t s t a r t u p. 2 P r e s s t h e M I S C S E T b u t t o n o n t h e L C D s c r e e n. A t t a c h t h e f i l t e r p a p e r o n C o n t a m i n a t i o n S h i e l d. P l a c e t h e p r e p a r e d C o n t a m i n a t i o n S h i e l d o n a p r o j e c t p a r t o f t h e l o w e r r i g h t s i d e o f t h e S y r i n g e b l o c k i n t h e i n s t r u m e n t. T h e a t t r a c t i o n o f tp hre o j e c t i n g p a r t t i g h t l y h o l d s t h e C o n t a m i n a t i o n S h i e l d. T h i n s e r t t w o h o l d i n g s h a f t s o n t o p o f t h e S y r i n g e b l o c k i n t o t h e h o l e s o n t P r o t e c t o r. P r e s s t Mh ie s c S e t b u t t o n a g a i n. 16

22 < M I S C S E T o n E x i P r e p 1 6 D x L C D p a n e l > < P l a c i n g t h e C o n t a m i n a t i o n S h i e l d o n t h e p r o j e c t i n g p a r t ( r e d a r r o w ) > 17

23 < I n s e r t i n g t w o h o l d i n g s h a f t s ( r e d a r r o w s ) i n t o t h e T i p P r o t e c t o r > 3 T h e p r e p a r a t i o n p r o c e s s i s a s f o l l o w s : C h e c k m a t e r i a l-> s B u f f e r C a r t r i d g e P r e p a r a-> t i op n r e p a r i n g t h e E l u t i o n TRuabce k -> T i p L o a d i n g -> C l i n i c a l S a m p l e L o a d i-> n g L o a d i n g t h e I n s t r u m e n t P. r e p t o o l s S e t u p T r a y H o l e P u n c h S a m p l e T u b e R a c k E l u t i o n T u b e R a c k D i s p o s a b l e T i p R a c k C. o n s u m a b l e s B u f f e r C a r t r i d g1 e s a n d 2 S a m p l e L o a d i n g T u b e s D i s p o s a b l e T i p s E l u t i o n T u b e s E l u t i o n T u b e C a p s P r o t e c t i o n C o v e r W a s t e T r a y 18

24 D i a g n o s t i c k i t t u b e s 4 5 P l a c e S e t u p t r a y o n a f l a t s u r f a c e d d e s k. O n t h e S e t u p t r a y, p l a c e Bt uh fe f e r C a r t r i d 1 g e, 2, D i s p o s a b l e t i p r a c ak n d E l u t i o n t u b e r a. c k 6 F i l l t h e a p p r o p r i a t e n u m b edr i so pf o s a b l e T i pisn t h e D i s p o s a b l e T i p R a. c k 7 I n s e r t a p p r o p r i a t e n u m b e r s o f t h e E l u t i o n T u b e s a n d D i a g n o s t i c K i t T u b e s i n t E l u t i o n T u b e R a. c k W e r e c o m m e n d m a r k i n g e a c h s t r i p o f t h e e l u t i o n s t r i p s w i t t h e c o r r e s p o n d i n cg o l u m n n u m b e r. 19

25 8 F a s t e n t h e P r o t e c t i o n C o v eorn t o t h e e l u t i o n t u b e r a c k L o a d i n g c o n t r o l s a n d s a m p l e s B. u f f e r c a r t r i d g e p r e p a r a t i o n 1 R e m o v e t h e s h r i n-w k r a p e n c l o s i n g b o t h t Bh ue f f e r C a r t r i d g1 e s a n d 2. : I f u s i n g a b ( l e a. d g e. k n i f e o r s c i s s o r s ), p l e a s e b e c a r e f u l n o t t o h u r t y o 2 C l e a n t h e s u r f a c e ( p r e f e r a b l y a p o s i t i v e p r e s s u r e b e n c h, e. g. c l e a n w h e r e w o r k w i l l b e p e r f o r m e d. : I n s p e c t t h e w e l l s o f B ut fh e f e r C a r t r i d g ae ns d m a k e s u r e a l l l i q u i d s a r e a t t h e b o t t o m o f t h e w e l l s. I f n e c e s s a r y, Bgur fa fb e r t h Ce a r t r i d gae ns d s n a p y o u r w r i s t t o b r i n g d o w n l i q u i d s t h a t m a y b e p r e s e n t o n t h e w e l l w a l l s. 3 4 R e m o v e t h e a c r y l l i d s fbruo fm f e r C a r t r i d g1 e s a n d 2. P u n c h h o l e s w i t h e t hh o l e P u n c h a c c o r d i n g t o s a m p l e n u m b e r. 20

26 5 C o v e r B u f f e r C a r t r i d g1 e s a n d 2 w i t h t h e a c r y l l i d s a f t e r h o l e p u n c h i n g i s c o m p l e t e S. a m p l e L o a d i n g 1 2 C l e a n t h e n e g a t i v e p r e s s u r e s u r f a c e ( e. g. bciaobsianfeet t) y o n w h i c h t h e n u c l e i c a c i d e x t r a c t i o n p r e p a r a t i o n w i l l b e p e r f o r m e d. T a k e o u t t h e n e c e s s a r y n u m b e r o f S a m p l e T u b e s a n d i n s e r t t h e m i n t o s e p a r a t e r a c k w i t h a t l e a s t 2 0 m m s p a c i n g b e t w e e n t h e h o l e s. 3 4 T a k e t h e o r i g i n a l c l i n i c a l sc ao mn tp la e i n e r s. D e -c a p t h e o r i g i n a l c l i n i c a l s a m p l e c o n t a i n e r s a n d p i p e t t e d L s a i n t o a S a m p l e L o a d i n g T u b e. 21

27 5 M o v e t h e f i l l e d S a m p l e T u b e i n t o S a tm hp e l e T u b e R a c k. : I n s e r t t h e S a m p l e T u b e s v e r t i c a l l y t o p r e v e n t s p i l l i n g. 6 D e -c a p a n o t h e r o r i g i n a l c l i n i c a l s a m p l e c o n t a i n e r a n d p i p e t t e d L s a i n t o t h e n e x t a v a i l a b l e s a m p l e l o a d i n g t u b e. 22

28 7 R e p e a t s t e p s 4 ~ 6 u n t i l a l l s a m p l e s a r e l o a d e d. : I f f o r a n y r e a s o n g l o v e c o n t a m i n a t i o n b y s a m p l e i s s u s p e c t e d, i m m e d i a e x c h a n g e g l o v e s t o p r e v e n t c o n t a m i n a t i o n o f s a m p l e s. 8 P l a c e t h e S a m p l e T u b e R a c k a n d W a s t e T r a y o n t o t h e S e t u p T r a y L. o a d i n g t h e I n s t r u m e n t 1 O p e n t h e d o o r o f t he e x i P r e p 1 6 D x a n d p u l l t h e B a s e P l a t e o u t c o m p l e t e l y. 2 S t a r t i n g f r o m t h e B u f f e r C a r t r i d g e s, p l a c e e a c h c o m p o n e n t o n e -b y -o n e i n t o t h e B a s e P l a t e a s d e s c r i b e d b e l o w. 23

29 ( 1 ) P l a c e t h e B u f f e r C a r t r2 i d goe n t h e h e a t i n g b l o c k o f t h e b a s e p l a t e. : I f B u f f e r C a r t r 2 i d g ie s n o t p r o p e r l y p l a c e d o n t h e h e a t i n g b l o c k, i t m a y c a u s e a n e x p e r i m e n t f a i l u r e o r a n i n s t r u m e n t m a l f u n c t i o n. ( 2 ) P l a c e t h e B u f f e r C a r t r1 i d go e n t h e b a s e p l a t e. : P l a c e t h e B u f f e r C a r t r 1 i d g e b y p u t t i n g t h e c a r t r i d g e t o t h e l e f t s i d e o f t h e b a s e p l a t e a n d p r e s s r i g -h h ta n d s i d e o f t h e C a r t r i d g e t o p r e s s t h e C a r t r i d g e t o s e c u r e. ( 3 ) P l a c e t h e S a m p l e t u b e r a c k o n t h e b a s e p l a t e. : B e c a r e f u l t h a t t h e S a m p l e t u b e r a c k s f r o n t a n d r e a r s i d e s a r e p r o p e r l y p l a c e d. 24

30 ( 4 ) P l a c e t h e W a s t e t r a y i n b e t w e e n t h e S a m p l e t u b e r a c k a n d t h e B u f f e r C a r t r i d g e 2. ( 5 ) P l a c e t h e E l u t i o n t u b e r a c k o n t h e b a s e p l a t e. C h e c k t h e P r o t e c t i o n C o v e r i s p r o p e r l y s e c u r e d o n t h e e l u t i o n t u b e r a c k. ( 6 ) P l a c e t h e D i s p o s a b l e t i p r a c k o n t h e b a s e p l a t e. : M a k e s u r e t h a t t h e t i p s, h o l e s a n d t u b e s a r e i n a l i g n m e n t. 3 R e m o v e t h e a c r y l l i d s f r o m t h e B u f f e r C a r t r i d g e s. 25

31 4 S l i d e t h e B a s e p l a t e b a c k i n t o t h e i n s t r u m e n t a n d c l o s e t h e E d x o i o P r e p o f 1 6 t hd e x. : M a k e s u r e t h a t t h e a c r y l l i d s o f t h e B u f f e r C a r t r i d g e s a r e r e m o v e d a c o m p o n e n t s a r e i n t h e c o r r e c t p o s i t i o n s. I f u n u s e d w e l l s a r e p r e s e na t c, r y l p l a c e t l i d s f a c e d o w n o n t o p o f t h e i n s t r u m e n t u n t i l s a m p l e p r e p i s c o m p l e t e R u n n n i n g t h e E q u i p m e n t 1 P r e s s t h e S T A R T b u t t o n t o a c c e s s P tr he e P S E T U P s c r e e n. 2 R e f e r t o t h e L i s t o f p r o t o c o l n u m b e r s i n t h i s K i t M a n u a l tth o r e se-d e li eg ci t t h e p r o t o c o l n u m b e r a p p l i c a b l e t o y o u r d e s i r e d n u c l e i c a c i d a n d s a m p l e s o u r c e t y p 3 4 V e r i f y t h e P r e p T y p e a n d S a m p l e S R C o f t h e t h-d r ei eg i t p r o t o c o l n u m b e r y o u h a v e e n t e r e d. P r e s s t h e E n t e r b u t t o n t o a c c e s s Et lh ue t i o n V o l u m e s e l e c t i mo en n u. 26

32 5 6 S e l e c t t h e e l u t i o n v o l u m e a n d p r e s s O K t h e b u t t o n t o c o m p l e t P e R E P S E T U P. C h e c k t h a t a l l r a c k s a n d B u f f e r C a r t r i d g e s a r e p l a c e d i n t h e i r r e s p e c t i v e o n t h e B a s e P l a t e a c c o r d i n g t Co H te hc e K L I S T o n t h e L C D t o u c h s c r e e n a n d P r e s s t h e O K b u t t o n t o i n i t i a t e t h e e x t r a c t i o n p r o c e s s. 7 V e r i f y t h e n a m e s o f t h e t a r g e t n u c l e i c a c i d t y p e a n d s a m p l e s o u r c e t y p e o n R u n n i n g M o d e s c r e e n, a n d p r e s s t h e R U N b u t t o n. 8 A f t e r t h e e x t r a c t i o n r u n i s c o m p l e t e, p u l l o u t t h e B a s e P l a t e c a r e f u l l y a n d r e m o v e t h e B u f f e r C a r t r i d g e s a n d a l l r a c k s f r o m t h e B a s e P l a t e. A f t e r r e m o v i a c c e s s o r i e s, p u s h t h e B a s e P l a t e b a c k i n c o m p l e t e l y a n d c l o s e t h e d o o r. 27

33 9 P r e s s t h e S T A R T b u t t o n t o i n i t i a t e U V s t e r i l i z a t i o n. T h e s t e r i l i z a t i o n p r o c e 5 m i n u t e s. P r o g r e s s c a n b e c h e c k e d t h r o u g h t h e p r o g r e s s b a r. 10 R e m o v e t h e c o n t a m i n a t i o n p r o t e c t i o n a c c e s s o r i e s ( T i p p r o t e c t o r, C o n t a m i n a t i s h i e l d ) P r e p a r i n g f o r f o l -u l op w w o r k I n o r d e r t o l o g i c a l l y p r o c e s s t h e s a m p l e s f re ox m i P ur p e T p M t o 1 6 D x w i t h o u t c o n f u s i n g t h e o r d e r o r i d e n t i t y o f s a m p l e s, p l e a s e f o l l o w t h e i n s t r u c t i o n s b e l o w (- p e r f o r m s t e r i l i z e d s t-p a tr ie cs s u r e e n v i r o n m e n t ) : 1 O p e n t h e d o o r o f t he e x i P r e p 1 6 D x a f t e r t h e n u c l e i c a c i d e x t r a c t i o n p r o c e s s i c o m p l e t e, a n d r e m o v e t hee l u t i o n T u b e R a. c k 2 U s e t h e P r o t e c t i o n C o v e r S e p a r a t i o n T o o l t o r e m o v e t h e P r o t e c t i o n C o v e ra s f o l l o w s. 28

34 : W o r k w i t h t hp e r o t e c t i o n C o v e r S e p a r a t i o n Tgoeonlt l y s o t h e c o v e r d o e s n o t p o p. ( 1 ) P l a c e t h e E l u t i o n T u b e R a cfkr o m E x i P r e T p M 1 6 D x o n t h e P r o t e c t i o n C o v e r S e p a r a t i o n T o oc al r e f u l l y. ( 2 ) P l a c e y o u r h a n d s o n tshee p a r a t i o n T o o al s a p i c t u r e. 29

35 ( 3 ) A p p l y g e n t l e p r e s s u r e o n y o u r t h u m b s a n d o t h e r f i n g e r s a n d g e n t l y p r e s s d o w n o n t h e S e p a r a t i o n T o o. l ( D o n o t p r e s s t h e P r o t e c t i o n C o v e r.) : I t h e l p s t o w i g g l e t h e S e p a r a t i o n T o o l t o f a c i l i t a t e s e p a r a t i o n o f t h e t u b e s f r o m t h e p r o t e c t i v e f i l m w i tn hs ie n r t t h e P r o t e c t i o n C o v e r. ( 4 ) C h e c k t h e P r o t e c t i o n C o v ear n d m a k e s u r e t h a t t u b e s a r e n o t s t u c k t o t h e P r o t e c t i o n C o v e. r 3 C a p t h e e l u t i o n t u b e s w i t h t h e s u p p l i e d E l u t i o n T u b e c a p s a n d p e r f o r m a b s p i n-d o w n. O b s e r v e t h e l e v e l o f l i q u i d w i t h i n t h e e l u t i o n t u b e s. T h e l e v e l o f a l b e i t s m a l l, s h o u l d b e e v e n. M a k e n o t e o f s a m p l e s t h a t h a v e u n e v e n a m o u n t s o f l i q u i d, t a s m aiy b e i n d i c a t i v e o f e r r o n e o u s s a m p l e e x t r a c t i o n. E x c l u d e t s a m p l e s f r o m f u r t h e r a n a l y s i s a-p n ed r fr oe r m t h e e x t r a c t i o n p r o c e s s. 4 R e m o v e a l l c o n s u m a b l e s a n d c o m p o n e n t s f r o m t h e i n s t r u m e n t, s t a r t i n g w i t h t h B u f f e r C a r t r i d g e s a n d v a r i o u s r a c k s, a rn d ad li l s c l i q u i d s a n d c o n s u m a b l e s i n t h e i r a p p r o p r i a t e c o n t a i n e r s. : I f -uu ns e d w e l l s a r e p r e s e n t i n t h e B u f f e r C a r t r i d g e s, w i p e t h e f i l m s u r f a c e B u f f e r C a r t r i d g e s w i t h -f a rleie n tc l o t h o r w i p e w e t w i t h 7 0 % e t h a n o l. R e p l a c e t h e a c r l i d s o n e t h B u f f e r C a r t r i d g e s a n d k e e p t h e m i n a p o s i t i v e p r e s s u r e e. g.c b el ne ca h n ( 30

36 b e n c h ) u n t i l l a t e r u s e. 5 P r e s s t h e M i s c S e t b u t t o n, r e m o v e t h e T i p P r o t e c t o r a n d C o n t a m i n a t i o n S h i e l a n d p r e s s t h e M i s c S e t b u t t o n a g a i n. 6 P u s h t h e B a s e P l a t e b a c k i n a n d s h u t t h e i n s t r u m e n t d o o r. t h e n t h e U V s t e r i l w i l l b e g i n a u t o m a t i c a l l y. 31

37 1 2. L i s t o f p r o t o c o l n u m b e r s N o. P r e p T y p e S a m p l e s o u r c e G e n o m i c D N A W h o l e b l o o d G e n o m i c D N A A n i m a l t i s s u e G e n o m i c D N A F F P E t i s s u e G e n o m i c D N A P l a n t t i s s u e G e n o m i c D N A P l a n t s e e d G e n o m i c D N A R i c e G e n o m i c D N A C u l t u r e d c e l l G e n o m i c D N A G r a m ( + ) b a c t e r i a G e n o m i c D N A G r a m (-) b a c t e r i a G e n o m i c D N A Y e a s t G e n o m i c D N A F u n g i G e n o m i c D N A P l a s m a G e n o m i c D N A S e r u m G e n o m i c D N A B u f f y c o a t G e n o m i c D N A S p u t u m G e n o m i c D N A B A L G e n o m i c D N A S a l i v a G e n o m i c D N A S w a b G e n o m i c D N A U r i n e G e n o m i c D N A S t o o l G e n o m i c D N A C e l l f r e e b o d y f l u i d G e n o m i c D N A P l e u r a l f l u i d G e n o m i c D N A C S F G e n o m i c D N A E P S 32

38 1 2 5 G e n o m i c D N A R e s p i r a t o r y s a m p l e G e n o m i c D N A A m n i o t i c f l u i d G e n o m i c D N A F o r e n s i c s a m p l e G e n o m i c D N A B o n e m a r r o w G e n o m i c D N A B o n e G e n o m i c D N A D r i e d b l o o d s p o t G e n o m i c D N A S o i l 1 3. T r o u b l e s h o o t i n g L o w y i e l d obf a c t e r i a l g e n o m i c D N A D i d y o u a d d s u f f i c i e n t a m o u n t o f s a m p l e s? T h e y i e l d i s d e p e n d e n t o n t h e s a m p l e t y p e a m o u n t. S o m e t i m e s o v e r l o a d s a m p l e m a y d e c r e a s e t h e y i e l d. D i d y o u c o m p l e t e l y l y s i s t h e s a m p l e s? I n c o m p l e t e l y s i s d e c r e a s e s t h e y i e l d a n d p u r i t D i d y o u s h a k e y o u r Bf ue fr c a r t r i d 2 g e b e f o r e u s e? I n c o m p l e t e s u s p e n s i o n o f t h e m a g n e t i c b e a d m a y d e c r e a s e t h e y i e l d a n d p u r i t y. C o -e l u t e d m a g n e t i c p a r t i c l e S o m e t i m e s m a g n e t i c p a r t i c l e -eclou t e d w i t h y o ubr a c t e r i a l g e n o m i c D N Aa f t e r B a c t e r i a l g e n o m i c D N A e x t r a c t i o n. -e C ol u t e d m a g n e t i c p a r t i c l e c a n n o t Bbaicntd e r i a l g e n o m i c D N A i n e l u t i o n b u f f e r a n d i t w i l l n o t d e c r e a s e t h e y i e l d a n d p u r i t y. 33

39 C o -e l u t e d m a g n e t i c p a r t i c l e s c a n e a s i l y s e p a r a t e b y s i m p l e c e n t r i f u g a t i o n R e m a r k s P l e a s e s t o r e t h e n u c l e i c e xa tc ri ad c t i o n k i t p o r t i o n a t r o o m t e m p e r a t u r e. D o n o t r e u s e r e a g e n t s a n d t a k e c a r e n o t t o m i x u p u s e d a n d f r e s h r e a g e n t s. P l e a s e u s e g l o v e s a n d w e a r a m a s k w h e n h a n d l i n g t h e c l i n i c a l s a m p l e s, a n d s t e r i l i z e d f i l t e r t i p s f o r a l l p i p e t t e s t e p s R e f e r e n c e s O b a t a, K., e t a l ( ). D e v e l o p m e n t o f a n o v e l m e t h o d f o r o p e r a t i n g m a g n e t i c p a r t i c l m a g t r a t i o n t e c h n o l o g y, a n d i t s u s e f o r a u t o m a t i n g n u c l e i c a c i d p u r i f i c a t i o n. J. B i o e n g., 9 1 ( 5 ), H o n o r e -B o u a k l i n e., e t a l ( ). R a p i d D i a g n o s i s E x ot fr a p u l m o n a r y T u b e r c u l o s i s b y P C R : I m p a c t o f S a m p l e P r e p a r a t i o n a n d D N A E x t r a c t i o n. J. C l i n. M i c r o b -2 i o3l : B o o m, R., e t a l ( ) I m p r o v e d s i l-gi uc a n i d i n i u m t h i o c y a n a t e D N A i s o l a t i o n p r o c e d u r e b a s e d o n s e l e c t i v e b i n d i n g o f b o v i n e a l p n h a ct ao s e si i l i c a p a r t i c l e s. J o u r n a l o f C l i n i M i c r o b i o l o g y 3 7, E x p l a n a t i o n o f s y m b o l s C a t a l o g N u m b e r C o n t a i n s s u f f i c i e n t f o r ( n ) t e s t s U S E B Y T e m p e r a t u r e L i m i t a t i o n 34

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