Thermo Fisher Scientific GeneJET RNA Purification Kit User guide

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PRODUCT INFORMATION

Thermo Scientific

GeneJET RNA Purification Kit

#K0731, #K0732



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www.thermoscientific.com/onebio

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#K0731, #K0732

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

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CERTIFICATE OF ANALYSIS

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  !"#$% &'(((#$)

 (((*  (#$$

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#$(/99:%8 

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1=%<$##<9

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#$%@1618$1<#%<<8<+

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$6C1#6<=$1<#/3

6#8/3

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2

COMPONENTS OF THE KIT

GeneJET RNA Purification Kit 50 preps

E&)39/

250 preps

E&)39

%& +))F A

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G4 (

7

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#$% 8(

7

(*

8((  A) A)

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STORAGE

%&(($(

7)H8< :(/A7AH8

Note. Close the bag with GeneJET RNA Purification Columns tightly after each use!

DESCRIPTION

!"I#$% &(  (#$

( ( ((44( ((4 4 

:(J( 7 ( 5 (4

(  (4( (  (7

( 

#$( (()) ( (*!"#$% 

&/A(7'(#$ *

*(  (7%847'%84#(#$7

(See(/ ((#$(5 



PRINCIPLE

((JB4*   4

 ( ( #$#/(

*((  (  (( 

#$( *(( (

'(4 5(5* (*

*%#$((*  * (7

*

3

Table 1. ((#$(5 



Source Quantity Yield, µg

=



)



/)

7

/A



=

 (



9)



-

7

/)



=( 9) A79)

=:



9)



A

7

9)



=(5



9)



+)

7

+A



=( A /)7/A

Bacillus pumilis (( /

×

/)

0

 (( /A7)

Escherichia coli (( /

×

/)

0

 (( A79)

> (( A

×

/)

+

 (( 9A7D)

": (( A

×

/)

+

 (( D)7A)

83 (( /

×

/)

+

 (( )7A

Saccharomyces cerevisiae (( D

×

/)

-

 (( /A)7/+)



IMPORTANT NOTES

Buffer preparation and handling

$ 5((0+7/))KWash Buffer 1  

Wash Buffer 2  L



50 preps

E&)39/

250 preps

E&)39



Wash Buffer

1

Wash Buffer

2

Wash Buffer

1

Was

h Buffer

2

8 * D) 9 )) /))

Ethanol (96

-

100%)

10

mL

3

9

mL

50

mL

170

mL

(5(



A)





+







A)





3)





$(4:  :(M 5 

(



• B #$ 4('Lysis Buffer

*N7 ($)F/D9=N7 ()F=

 /5(Lysis Buffer

• 8 :Lysis Buffer(  7(5 

*(93H84 ( :*AH8

• G(5*(Lysis Buffer and Wash Buffer 1 ( 

see /3$6C1#6<=$1<#(  *:4(

*((*

4

Avoiding ribonuclease contamination

#$(*( #$ 

#$4* (( (58

: #$#$4 (( (**

GB(

!( 5# L

• G(5*(#$(4:  

#8(5'(

• @(4(#7

• @5# 7(

4 *: 

• &((: ((*$ (((



Starting material handling and storage

• G#$(4( ( (

5% (J' :((

• G(  ( 54(*

( (( ('(#$

• 1#$( (( 4J(('

73)H86J((((**

(*

• $( ( (( ((73)H8('>*54

#$  ((J (4((5( 



• B(( (( #$ ( ((( *

((DH8(J((



5

ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED

• %(4#7

• O

• =  

• =  

• ((5

• '(J L

o =(

o 7J

o B((

• /D9=N7 (=

• (0+7/))K



Buffers

• 6( ( (((L

%B/93=#8(43=&8(4/)=#



>%<

D

4=&>



%<

D

4>3D

• 67575 (L

/)=>8(4>-)4/=$ (J)D,(

 

• 6(L

C(/=(4)/=$4>3D$)/K7N7 (

A)( J()P

• 6%&(L

/)=>8(4>-)4/=$



6

RNA PURIFICATION PROTOCOLS

% (#$ ( ( ((44 4(4

  +7/9



A. Mammalian Tissue and Insect Total RNA Purification Protocol

Before starting:

• ('B*N7 ($)F

/D9=N7 (= /5(B'

• %'%&(L(/)F%& (

A0)F/)=>8(4>-)4/=$



Step

Procedure

1

G9)J

4: 





(((*L

 (

%( 9)/)( ('

(*( 

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9))QB(*N7 (O/)

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•  *B' :((

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•  ( (#$ (



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(*((  #$

>J(7J(

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 J7J

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2

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Note. Close the bag with GeneJET RNA Purification Columns tightly after each

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#$% 8( /./)))× 

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./)))×

Optional1((  (4 (( 

7 (/

  ((  (*7(

!"#$% 8((/A#7

  

 (

9

$

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G4 (

7

 ( !"#$

% 8(8/./)))×(#$

Note

• =0)K#$((6#$

((*(/))QG4 (7

• 1  #$'#$(((

(4RA5(G4 (7

  

A)Q

 (%(*((5(

((((('(#$

10

   (@#$*( 

#$7)H873)H8(

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8

B. Mammalian Cultured Cells Total RNA Purification Protocol

Before starting:

• ('B*N7 ($)F

/D9=N7 (= /5(B'

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Step

Procedure

1



 ((

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%((/×/)

3

 ((  A

A)×  (( *%B5

(*  

$ ((

5* ((A×/)

+

 (( ((

 *%B5(5 %B 

(( (( 5(%B

J ((   (((

 AA)× 

Note

%(( ((  (#$(

7

3)H8(

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2



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 (( (( //

+))Q(B

(*N7 (O/)(

Note

• 1B 5 4

J('>J(9)

7J(()7

(

#75(1 (J*(((

  #$(

• 1 ((5(4 A

/D)))×*#7  

 (

3

$9+)Q

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(0+

7

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

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3))Q(!"#$% 8(

 (( 8 (/./)))× 

(*7(   ( : (( 

(((( (

  ((  (*7(%( 

!"#$% 8(* ((  (

Note. Close the bag with GeneJET RNA Purification

Columns tightly after each

use!

5

$3))QGB/(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

9

Step

Procedure

6

$+))QGB(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

7

$A)Q



GB!"#$% 8(



./)))×

Optional1((  (4 (( 

7 (/

  ((  (*7(

!"#$% 8((/A#7  

 (

8

$/))QG4 (7 ( !"#$

% 8(8/./)))×(#$

Note

• =0)K#$((6#$

(4(*(/))QG4 (7

• 1  #$'#$(((

(44S/×/)

+

 ((45(G4 (7



 A)Q (%(*

((5(((((('(#$



9

 

  (@#$*( 

#$7)H873)H8(



10

C. Human Blood Cells Total RNA Purification Protocol

Before starting:

• ('B*N7 ($)F

/D9=N7 (= /5(B'

• B(( (( #$ ( ((( *

((DH8(J((



Step

Procedure

1

8(( ( (( )A*((D))

×

A

DT8B( ((*(((((+)73)K((

5(5 ((((*

2

((+

))Q





B(*



N7 (O(

3 $DA)Q(0+7/))K

4



3))

Q





(!"#$% 8(

 (( 8 (/./)))× (*7

(   ( : (( 

(((( (

  ((  (*7(



%( 

!"#$% 8(* ((  (

Note. Close the bag with GeneJET RNA Purification

Columns tightly after each

use!

5

$3))QGB/(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

6

$+))QGB(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

7

$A)QGB!"#$% 8(

./)))×

Optional1((  (4 (( 

7 (/

  ((  (*7(

!"#$% 8((/A#7  

 (

8

$A)Q

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G4 (

7

 ( !"#$

% 8(8/./)))×(#$

9

   (@#$*( 

#$7)H873)H8(



11

D. Bacteria Total RNA Purification Protocol

Important Note

6#$(  (((5(*

<

+))

U)A7/Do not use an overnight culture for RNA isolation.



Before starting:

• ('/)=>8(4>-)4/=$*

(J ((  )D,

• ('B*N7 ($)F

/D9=N7 (= /5(B'



Step Procedure

1

/A!75!75 ( (/

×

/)

0



((/A  8((  (( 

./)))8((54(5(((

2 ((/))F((*

(J)D,(  155(

3

1  ((A

/A

7

A



8



4

$9))Q

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

B(*

N

7

 (





=

(5/A(

5

$/-)Q



(0+

7

/))K







6



3))

Q



(!"#$% 8(

 (( 8 (/./)))× (*7

(   ( : (( 

(((( (

  ((  (*7(%( 

!"#$% 8(* ((  (

Note. Close the bag with GeneJET RNA Purification Columns tightly after each

use!

7

$3))Q



GB/(*(4

9

!

"

#$% 8( /./)))× (*7

(   ( : (( 

8

$+))Q



GB(*(4

9

!"

#$% 8( /./)))× (*7

(   ( : (( 

9

$A)Q



GB!"#$% 8(

./)))×

Optional1((  (4 (( 

7 (/

  ((  (*7(

!"#$% 8((/A#7  

 (

12

(continued)

Step

Procedure

10

$/))QG4 (7 ( !"#$

% 8(8/./)))×(#$

Note

• =0)K#$((6#$

((*(/))QG4 (7

• 1  #$'#$(((

(4S/×/)

+

 ((5(G4 (7 

 A)Q (%(*((

5(((((('(#$

11

   (@#$*( 

#$7)H873)H8(



E. Yeast Total RNA Purification Protocol

Important Note

6#$( (((5(*

<

+))

U)A7/Do not use an overnight culture for RNA isolation. 6 ((

J ( (*((5 ((

Before starting:

• %C(L/=(4)/=$4>3D"

)/Kβ7 (A)( J()

• ('B*N7 ($)F

/D9=N7 (= /5(B'

• %'%&(L(/)F%& (

A0)F/)=>8(4>-)4/=$



Step

Procedure

1

 (( (D

×

/)

-

 ((/A  

58((  (( ./)))× 



2





 ((((/))

F





C(





3

1 9)H89)



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(   ( : (( 

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Note. Close the bag with GeneJET RNA Purification Columns tightly after each

use!

9

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10

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11

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./)))×

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  

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

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Note

• =0)K#$((6#$

((*(/))QG4 (7

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13

   (@#$*( 

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14

ADDITIONAL PROTOCOLS

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Genomic DNA Removal from RNA Preparations

A. Using DNase I, stand-alone product (#EN0521)

Step

Procedure

1

$#

7

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%87GE)+)/ /)Q

1 93H89)

Note.    :#1E<)9-/4 ((/,Q4 

 ( 5#$

2

$/QA)=$ +AH8/) 5#1

$$'#$(J*5( 

  (

$(5(4#$!"#$% &see/A#$

8(% ((, ( 

3

@#$*( 



Note

• /#1/Q#$

• O( A)=$(  ((

#$ (  #$)/Q,Q



B.Using Thermo Scientific RapiOut DNA Removal Kit (#K2981).

: 55(#$#$('5(

#1(*7 &  #14#7

#5( #15(

6(4(5***   ,



15

RNA Cleanup Protocol



!"I#$% &  (/))µ#$ (#1

J  



Step

Procedure

1

$P5( 

/))

F



*

G4 (

7



 ($9))FBwithoutN7 (=

(5

2 $/-)Q(0+7/))

%



3



!"



#$% 8( (( 

8 (/./)))×  (( 

(*7(%( !"#$% 8(

* ((  (

Note. Close the bag with GeneJET RNA Purification Columns tightly after each

use!

4

$3))QGB/(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

5

$+))QGB(*(4see9!"

#$% 8( /./)))× (*7

(   ( : (( 

6

$A)Q



G



B!"



#$% 8( 



./)))×

Optional1((  (4 (( 

7 (/

  ((  (*7(

!"#$% 8((/A#7

  

 (

7

$A)Q



G4 (

7

 (

 !"



#$

% 8(8/./)))×(#$

Note. 6#$((*(A)Q

G4 (7

8

   (@#$*( 

#$7)H873)H8(



16

TROUBLESHOOTING

Problem Possible cause and solution

Low RNA yield

Too much starting material was used for lysate preparation.

 ( ((

 ( (

Starting material was not completely disrupted.

 (1 

Ethanol was not added to the lysate.

=:(*(((

  (

Ethanol was not added to Wash Buffers 1 and 2.

=:(*GB/

6((* GB9

Degraded RNA

Inappropriate handling of starting material.

G#$(4( ( (

5% (J' :((

J(J('( (( 

73)H8*((5(

B

RNase contamination.

5# 4*(5 ((  

(5'(@(4(#75#

7(*: 

DNA

contamination

#$*#1E

#)A/



1 5#1

  (4(, ( 

7#$((*#$8(% (see/A

Column

clogging

Too much starting material was used for lysate preparation.

 ( ((

 ( (

Starting material was not completely disrupted.

 ( 

Tissue debris was not removed before applying lysate on a column

=(1 (#$% % (

8J ((5

(( (

Enzymatic

reactions not

running well

Ethanol was carried over into the eluted RNA.

1((  (**G

B4 (( 7 ((/

./)))×

Salt was carried over into the eluted RNA.

@GB  $(**  (*

GB/ **GB

17

SAFETY INFORMATION

Lysis Solution

>J7 ((LGuanidinium thiocyanate

Xn>(



Risk phrases

 >(*((*

9+,9- 1:

Safety phrases

9 ,,5,

+ 1   *4(*(*: (5 

9+,93 G( 5 ((5

+) ( J*



Proteinase K

>J7 ((LProteinase, Tritirachium album serine

Xn >(

Risk phrases

D = J(

Safety phrases

9 ,,5,

9+ G( 5 (

DA 1  (*((4: (5 (*((*

(

+) ( J*



References

/ 8 J:4%#  /0-3(7#$(  7

(7 ( $(B /+L/A+7/A0

 B448"$(4===(48"4%=G((4"5#/00)

(  (  "8(= (-LD0AWA)9



PRODUCT USE LIMITATION

 5(4( (5( in vitro ( 

* 5(4(

(

%(***   ,=( 

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X)/D6  1 $((5$((:6

  1 

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Thermo Fisher Scientific GeneJET RNA Purification Kit User guide

Thermo Fisher Scientific GeneJET RNA Purification Kit User guide

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