Coller Protocol Book

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The Coller Lab Protocol Book
Revised September 6th, 2012
Table of Contents
...........................................................................................................
Solutions (gel mixes)

6
...................................................................................
10 % Ammonium Persulfate (APS)

7
...........................................................................
Ampicillin (100 mg/mL) Stock Solution

7
.......................................................................................
BSA (10 mg/mL) Stock Solution

8
............................................................................................................
1 M calcium chloride

9
....................................................................................................
Chruch Buffer (modified)

9
.....................................................................................................
Church Wash (modified)

9
.................................................................................................
Competent Cell Protocol

10
.............................................................................................
Denhardt’s Solution (100X)

11
...........................................................................
DEPC Treated Water (for RNA work)

12
............................................................................................................
10% DepC Solution

13
..............................................................................................................
Dilutor A Solution

13
..........................................................................................
Double distilled H2O (sterile)

13
..................................................
0.5 M EDTA pH 8 (ethylene diamine tetra-acetate)

14
..........................................................................................................................
1M Glucose

14
....................................................................................
IPTG Stock Solution (0.12 g/mL)

15
..............................................................................................................................
LB Media

16
.................................................................................................................
LB Media Plates

16
.............................................................................................................................
M9 media

17
.................................................................................................
M9 Minimal Media Plates

17
.............................................................................................................................
M9 Plates

18
.......................................................................................................................
10 x M9 salts

18
...................................................................................................
1M magnesium chloride

19
..........................
1M MgCl2 (Magnesium chloride), 1M MgSO4 (Magnesium sulfate)

19
....................................................................................................
Neutralization Solution

20
..............................................................................................................................
10x MMR

20
............................................................................................................................
20x MOPS

21
...................................................................................
Pen-Strep 1000 X Stock Solution

21
...............................................................................................................
Phosphate Buffer

22
..............................................................................
1x PBS (Phosphate-Buffered Saline)

23
................................................................................................................................
10x PBS

23
....................................................................................................
Pipet Cleaning Protocol

23
.....................................................................................................
1 M potassium chloride

25
.................................................................................................
Proteinase K (10 mg/mL)

25
.............................................................................
RNase A Stock Solution (10 mg/mL)

26
.........................................................................................................
Silanized Glass Slides

27
..................................................................................
10% SDS (sodium dodecyl sulfate)

28
.....................................................................................
SDS-Page Running Buffer (10X)

29
....................................................................................
SDS-Page Separating Buffer (4x)

29
........................................................................................
SDS-Page Stacking Buffer (4x)

29
...................................................................................................
SDS-Page Stain/Destain

30
................................................................................................
SDS Gel Loading Dye (2X)

30
..............................................................
20 x SSC (Sodium Chloride / Sodium Citrate)

30
................................................................................................
3M sodium acetate pH 5.2

31
...........................................................................................................
5M sodium chloride

31
...........................................................................................................................
SOB media

32
.....................................................................................................................
Soft Top Agar

33
......................................................................................
25% sucrose, 50 mM Tris pH 7.5

33
................................................................................................................................
1 M Tris

34
...................................................................
5 xTBE Buffer (Tris-Borate-EDTA Buffer)

35
....................................................................................................................
1 x TBE Buffer

35
..............................................................................................................................
10 X TBE

35
.......................................................................................
10x TBS (Tris-Buffered Saline)

36
................................................................
TYE plates  Tryptone Yeast Extract Media

37
............................................................................................
5x Western Transfer Buffer

38
......................................................................................................
X-GAL Stock Solution

38
............................................................................
SD media Drop-Out Amino Acid Mix

39
.......................................................
Yeast Liquid Media: SD, SG, or SR Minimal Media

40
...................................................................
Yeast Plates: SD, SG, or SR Minimal Media

40
............................................................
YPD Media  Yeast Peptone Dextrose Media

41
..............................................................
YPD Plates  Yeast Peptone Dextrose Plates

41
..............................................................................................................
Sporulation Media

42
..........................................................................................
Preparation of DNA markers

43
........................
Protocol A2 Preparation of Heat Shock Competent Cells (HB101)

44
..........................................................
Transformation of frozen Competent Bacteria.

45
...........................................................................................
QUICK TRANSFORMATION

45
..............................................................................................................
Restriction Digest

46
Small-
scale
...............................................
Isolation of Plasmid DNA from Bacteria (Mini-preps)

47
....................................................................................
Analysis of DNA on Agarose Gel

48
..........................................................................................
SDS-Page Gel Electophoresis

49
...........................................................................
Sarnow Translational Extract Buffers

50
...........................................
Preparation of Yeast S30 Lysate for in vitro Translation

51
.....................................................................................................
Agarose Northern blot

53
.............................................................................................
DNase I Treatment of RNA

54
........................................
In Situ Hybridization of mRNA and Immunofluorescence

55
..........................
Kinasing Oligonucleotides for use as Probes in Northern Blotting

58
................
Synthesis of Random-primed DNA probes for Northern Hybridization

59
..................................................
Isolation of Plasmid DNA – Rapid Lysate Procedure

60
..............................................................................................
RNase H Digestion of RNA

62
......................................................................................
Yeast Glass Bead RNA Isolation

63
...................................................................................
Recipe for 6% polyacrylamide gel

64
............
Recipe for pre-hybe for Northern Blots probed with Radiolabelled Oligo

64
.....
Recipe for pre-hype for Northern Blots probed with Random primed probes

64
..............................................................
mRNA Decay Analysis – Galactose ‘Shut-off’

65
...............................................................................................
Primer Extension Analysis

67
.........................................................
Northern Blot Hybridization using a Riboprobe

69
............................................................................................................
Polysome Protocol

70
...................................................................
Growth Conditions for Polysome Analysis

71
......................................................
Polysome Lysis/Sedimentation Analysis Protocol

71
.................................................................................................
Polysome Buffer Recipies

73
......................................................................................................
Quicksite mutagenesis

75
...................
Reference: The ribosomal DNA (rDNA) of Saccharomyces cerevisiae

76
...............................................................................
Reference: RNA polymerase usage

76
.....................................................
Reference: Approximate Size of ribosomal RNAs

76
.......................................................................................
Calculation of mRNA half-lives

77
.........................................................................
Preparation of Competent E. coli Cells

78
..........................................................................................
Preparation and Use of G418

79
...........................................................................................
Tandem Affinity Purification

80
................................
Western Blotting of Proteins from Yeast Whole Cell Extracts

82
...........................................................................................
Pouring SDS PAGE Mini Gels

83
..........................................................................
Western blotting and Immunodection

84
..................................................................................................................
Immunodection

85
..............................................................................................
Yeast Immunoprecipitation

86
.................................................................................................................
GST purification

87
......................................................................................................................
pCp Labelling

88
........................................................................
TAP - Affinity Purification ala Wenqian

89
................................
Stripping proteins from Western blot membranes by Low pH

91
.................................................................................
Gradient Fractionator Parameters

92
.......................................
PCR Amplification of DNA from a Single Bacterial Colony

93
...............................................................................
Useful Electrophoresis Information

94
........................................................................
In vivo 35S labeling / TCA precipitation

95
...............................................................
Yeast Ribosome Transit Time Measurement

96
............................................................................................
Oligo dT Cellulose Selection

98
...................................................................................................................
Generating pCp

99
................................................
‘Quick Change’ Site-Directed Mutagenesis Protocol

100
....................................................................................................................
Anchor Away

102
.........................................
Splint Ligation RT-PCR for Detecting Decapped mRNA

104
Affinity Purification of FLAG Tagged Ribosome for Detecting mRNA Decay
...............................................................................................................................
Factors

107
...................................................................................................
Circularization RT-PCR

109
........................................................................................................
Poly(A) tailing assay

111
THE BASIC PRINCIPLES OF HOW ETHANOL PRECIPITATION OF DNA AND
....................................................................................................................
RNA WORKS

113
......................................................................................
Preparation of Dialysis Tubing

115
...............................................................................................
ECL Reagent: Homebrew

116
.....................................................................
YEAST GENOMIC DNA PREPARATION

117
................................................
RNA Extraction from Polysome Gradient Fractions

118
................................................................
Formaldehyde Cross-linking of Yeast Cells

119
.........................
Mapping single end reads in Galaxy (http://main.g2.bx.psu.edu/)

120
S
OLUTIONS
(
GEL MIXES
)
Acrylamide is used in the lab to make special types of electrophoretic gels which we can use to
separate very small pieces of nucleic acids. Acrylamide will polymerize when combined with
APS (Ammonium Persulfate) and another solution called TEMED. We add these two catalysts to
acrylamide and pour the solution between two glass plates (gel plates). When the acrylamide
polymerizes, if forms a gel with very small pores through which nucleic acids can travel. These
are referred to as polyacrylamide gels or PAGE (polyacrylamide gel electrophoresis).
WARNING: Polyacrylamide is a neurotoxin when liquid, harmless when polymerized.
Always wear gloves and avoid contact with skin. Safety glasses should also be worn.
20% Acrylamide Solution
!
!
!!!!500 mL
!10 X TBE !!!50 mL
!Urea!!!!210 g
!Acryl/Bis solution!!250 mL
!dH
2
O!!!!3 5 m L
!!M i x u n t i l d i s s o l v e d
!!D o n o t a u t o c l a v e
!!W r a p b o t t l e i n f o i l ( p h o t o r e a c t i v e )
!!S t o r e i n c o o l e r
6 % A c r y l a m i d e S o l u t i o n
!
!
!!!!1 L
!
2 0 % A c r y l a m i d e S o l u t i o n!3 0 0 m L
!D i l u t o r A!!!7 0 0 m L
!!M i x u n t i l d i s s o l v e d
!!D o n o t a u t o c l a v e
!!W r a p b o t t l e i n f o i l ( p h o t o r e a c t i v e )
!!S t o r e i n c o o l e r
##H e a t t h e 2 0 % a c r y l a m i d e s o l n fi r s t t o d i s s o l v e t h e c o n t e n t s
10

%

A
MMONIUM
P
ERSULFATE
(APS)
APS is used to assist in the polymerization of polyacrylamide. APS acts as a catalyst in this
reaction.
!!!!!!
5 0 m L
!A m m o n i u m P e r s u l f a t e!!! 5 g
!!D i s s o l v e i n w a t e r a n d b r i n g t o v o l u m e
!!A l i q u o t 5 0 0 µ L i n t o e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o d i s t i n g u i s h f r o m
!!!!o l d e r b a t c h i f p o s s i b l e.
!!S t o r e a t 4 ˚ C
M a k e a f r e s h b a t c h e v e r y m o n t h!
A
M P I C I L L I N
(100
MG
/
M
L)

S
TOCK
S
OLUTION
Ampicillin is a common laboratory antibiotic used to select for growth of bacteria which contain
plasmids which provide resistance to ampicillin's effects.
!!!!
5 0 m L !!!1 0 m L
!D r y A m p i c i l l i n!!5 g!!!1 g
!D i s s o l v e i n d H
2
O a n d d i l u t e t o fi n a l v o l u m e.
!M a k e 0.5 m L a l i q u o t s i n u n s i l a n i z e d e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o
!!!!d i s t i n g u i s h f r o m o l d e r b a t c h i f p o s s i b l e.
!L a b e l w i t h a n ‘ A ’ o n t h e l i d.
!F r e e z e
!D o n o t a u t o c l a v e, i t w i l l d e s t r o y a m p i c i l l i n.
BSA

(10
MG
/
M
L)

S
TOCK
S
OLUTION
BSA = Bovine Serum Albumin. BSA is generally used in the lab to provide a non-reactive protein
into a reaction or an experiment.
!!!!!
5 0 m L !!!1 0 m L
!!D r y B S A!!0.5 g!!!0.1 g
!D i s s o l v e i n d H
2
O a n d d i l u t e t o fi n a l v o l u m e.
!M a k e 1.0 m L a l i q u o t s i n u n s i l a n i z e d e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o
!!!!d i s t i n g u i s h f r o m o l d e r b a t c h i f p o s s i b l e.
!L a b e l w i t h a n ‘ B ’ o n t h e l i d.
!F r e e z e
!D o n o t a u t o c l a v e.
1

M
CALCIUM CHLORIDE
(MW 147.02)
!For 1 liter:
!!Dissolve 147.02 g of CaCl
2
•2H
2
O in 800 mL of dd water.
!!Adjust volume to 1 liter.
!!Dispense into 100 mL bottles
!!Autoclave on
liquid cycle
for 20 mins.
!Quantity needed:
!!It may be sufficient to make 500 mL at a time dependent on demand.
C
HRUCH
B
UFFER
(
MODIFIED
)
!For 500 ml:
!!16.75 g Na
2
HPO


7H
2
O
!!500 ul 85% Phosphoric Acid
!!1 ml 0.5M EDTA pH 8.0
!Heat to dissolve
!!35 g SDS
!Heat to dissolve
!!5 g BSA
!Heat to dissolve, either microwave for short bursts or stir and heat for several hours.
Store at 65º
C
HURCH
W
ASH
(
MODIFIED
)
!For 1 L:
!!11.17 g Na
2
HPO
4

7H
2
O
!!333 ul 85% Phosphoric Acid
!!2 ml 0.5M EDTA pH 8.0
!Heat to Dissolve
!!50 g SDS
!Heat to Dissolve
Store at 65º
C
OMPETENT
C
ELL
P
ROTOCOL
Media/Glassware
2L very clean flask which has not been exposed to detergents.
500 ml very clean flask which has not been exposed to detergents.
Sterile eppendorf tubes.
Sterile 200 ul Pipets.
1L plastic sterilization filter bottle.
TYE plate (or minimal media plate)
DMSO
KCl (250mM):
!For 100 ml, add 1.86g KCl to ddH2O
!Autoclave
MgCl2 (2M):
!For 100 ml, add 19 g MgCl2 to ddH2O
!Autoclave
SOB: In a 2L flask:
!!237 ml ddH2O
!!5 g Bacto-tryptone
!!1.25 g bacto-yeast extract
!!0.125 g NaCl
!Dissolve by shaking.
!Add!2.5 ml KCl (250mM)
!PH to 7.0 with NaOH (5M)~ approx. 50 ul
!Adjust volume to 250 ml
!Autoclave 20 min
Add 1.25 sterile MgCl2 (2M) to sterile flask just before inoculation.
!
TB: For 500 ml
!!1.51g H-Pipes
!!1.10g CaCl2
!!9.32g KCl
!pH to 6.7 with KOH. Then add 5.44 g MnCl2. Raise volume to 500 ml and filter sterilize
into 1L bottle.
Procedure:
Day 1: Streak out sells from a frozen stock onto a TYE plate. Grow O/N @ 37ºC (If competent
!cells will later be used to transform phage, cells should grow on minimal medium
!instead).
Day 2: Inoculate 250 ml SOB with 10-12 good sized colonies. Grow O/N at 18º with vigorous
shaking. (There is a cold shaker in the basement of Biochem.)
Day 3: Cells should be removed from shaker when OD600 is approximately 0.6. Place flask on
ice for 10 min. Transfer culture to the 500ml centrifuge pot and spin 10 min., 4ºC at 2500 x g.
Pour off supernatant. Resuspend pellet in 80 ml ICE-COLD TB. Incubate suspension on ice for
10 min. Spin suspension 10 min., 4ºC at 2500 x g. Pour off supernatant. Gently resuspend pellet
in 20 ml ICE-COLD TB. Add DMSO to a final concentration of 7 %. Incubate suspension on ice
for 10 min. Aliquot 200 ul cells into sterile eppendorf tubes. Quick freeze in liquid nitrogen.
Store at –80ºC.
D
ENHARDT

S
S
OLUTION
(100X)
!






5 0 0 m L

F i c o l l




1 0 g

p o l y v i n y l p y r r o l i d o n e


1 0 g

B S A




1 0 g
D i s s o l v e i n 3 0 0 m L o f w a t e r, b r i n g u p t o v o l u m e t o 5 0 0 m L. F i l t e r a n d d i s p e n s e i n t o 5 0 m L a l i q o u t s. S t o r e
a t - 2 0 º C





DEPC

T
REATED
W
ATER
(
FOR
RNA
WORK
)
!DEPC will destroy any RNAase present in a solution when autoclaved in its presence,
!therefore we use DepC water for any manipulations of RNA to prevent degradation.
WEAR GLOVES WHEN MAKING DEPC WATER (Two reasons)
!!
!!1 ] D E P C w a t e r i s u s e d f o r R N A w o r k a n d m u s t b e R N A a s e f r e e
!!2 ] D E P C i s a s u s p e c t c a r c i n o g e n a n d c o n t a c t w i t h s k i n s h o u l d b e a v o i d e d
P r o t o c o l:
!T r e a t 1 l i t e r o f d o u b l e d i s t i l l e d w a t e r w i t h 1 0 m L o f 1 0 % D E P C s o l u t i o n ( i n t h e
!r e f r i g e r a t o r i n t h e u n d e r g r a d r o o m ) a n d s t i r r o v e r n i g h t.
!T h e D E P C, w h i c h c a n b e h a r m f u l t o c h e m i c a l r e a c t i o n s a n d m o d i f y R N A m u s t t h e n b e
!d e s t r o y e d b y a u t o c l a v i n g t h e s o l u t i o n (
i n 1 0 0 o r 5 0 0 m L b o t t l e s: w h i c h h a v e p r e v i o u s l y
!b e e n b a k e d s u c h t h a t t h e y a r e R N A a s e f r e e )
t w i c e
f o r t h i r t y m i n u t e s. A f t e r
!a u t o c l a v i n g t h e w a t e r s h o u l d n o l o n g e r s m e l l o f D E P C. ( D E P C s m e l l s l i k e J u i c y F r u i t
!g u m )
Q u a n t i t y n e e d e d:
!T h e l a b s h o u l d b e s t o c k e d w i t h D E P C w a t e r o n a r e g u l a r b a s i s. W e s h o u l d a t l e a s t
!h a v e t e n b o t t l e s o f d e p c w a t e r a t a l l t i m e s.
S p e c i a l N o t e
!D u e t o t h e e x t e m e l y s e n s i t i v e n a t u r e o f R N A, p l e a s e, p l e a s e e n s u r e t h a t:
1. A t n o t i m e i n t h e p r o c e d u r e s h o u l d t h e D E P C w a t e r t o u c h a n y g l a s s w a r e w h i c h i s n o t b a k e d. 2.
N o r s h o u l d i t t o u c h a n y p l a s t i c w a r e w i t h t h e e x c e p t i o n o f d i s p o s a b l e p l a s t i c p i p e t t e s.
3. D o n o t l e a v e t h e D E P C w a t e r s i t t i n g u n c o v e r e d o n t h e b e n c h w h e r e i t c o u l d b e c o m e
c o n t a m i n a t e d.
O r a n g e c a p p e d b o t t l e s
! O r a n g e c a p p e d b o t t l e s w i l l o n l y b e u s e d f o r D e p C w a t e r. T h e l i d s w i l l b e l e f t o n t h e m
!w h e n t h e y a r e p l a c e d o n t h e w a s h t r o l l e y. P l e a s e s t o r e t h e m a n d w a s h t h e n i n b a t c h e s
!s e p e r a t e f r o m a l l o t h e r g l a s s w a r e. T h i s i s s o p r o t e i n s o r R N A s e s p o t e n t i a l l y p r e s e n t o n
!o t h e r g l a s s w a r e d o n o t c o n t a m i n a t e t h e s e b o t t l e s. T h e b e s t w a y t o p r e v e n t R N A s e
!c o n t a m i n a t i o n i s t o h a v e n o R N A s e s p r e s e n t r a t h e r t h a n t o a t t e m p t t o "k i l l" t h e
!R N A s e s b y b a k i n g. O n c e t h e y a r e w a s h e d t h e y s h o u l d b e b a k e d ( a n d t h e l i d s
!a u t o c l a v e d ) a n d s t o r e d i n t h e a s s i g n e d c u p b o a r d.
10%

D
EP
C

S
OLUTION
100% DEPC is supplied in 50 ml bottles. Add one of these bottles to 450 ml of ethanol in the fume
hood, mix throughly and store at 4 degrees in a 1 litre bottle. Leave the residual contents of the 50
ml bottle to evaporate in the fume hood before discarding the bottle.
D
ILUTOR
A

S
OLUTION
Dilutor A is a convenient solution used to dilute high concentrations of acryamide for running
lower percentage gel.
!
!!!!500 mL !!1 L
!5 X TBE !!!100 mL!!!200 mL
!Urea !!!!210 g!!!420 g
!dH
2
O!!!!2 3 5 m L!!!4 7 0 m L
!M i x u n t i l d i s s o l v e d
!D o n't a u t o c l a v e
!W r a p i n f o i l
!S t o r e i n c o o l e r
D
O U B L E D I S T I L L E D
H
2
O

(
S T E R I L E
)
D o u b l e d i s t i l l e d w a t e r ( d d H
2
0 ) s h o u l d b e c o l l e c t e d i n t h e c a r b o y s f r o m t h e R e z l a b n e x t d o o r.
T h e d i s t i l l a t i o n p r o c e s s r e m o v e s i m p u r i t i e s s u c h a s s a l t s f r o m t h e w a t e r, I T D O E S N O T s t e r i l i s e
t h e w a t e r. I n o r d e r t o s t e r i l s e i t i t m u s t b e a u t o c l a v e d a s d e s c r i b e d b e l o w:
!D e c a n t t h i s w a t e r i n t o 1 0 0 m L a n d 5 0 0 m L b o t t l e s a n d l a b e l t h e m.
!A u t o c l a v e f o r 2 0 m i n s o n
l i q u i d c y c l e
.
!Q u a n t i t y n e e d e d:
!!1 0 b o t t l e s, 1 0 0 m L e a c h s h o u l d b e s u f fi c i e n t t o l a s t t h e l a b a b o u t f o u r d a y s.
!!R o u n d b o t t l e s p r e f e r r e d t o t h e s q u a r e b o t t l e s.
0.5

M

EDTA
P
H

8


(
ETHYLENE DIAMINE TETRA
-
ACETATE
)

!!!
M W 2 9 2.2 5 g
E D T A i s a c h e l a t o r o f d i v a l e n t c a t i o n s, w h i c h a r e n e c e s s a r y f o r m a n y b i o l o g i c a l r e a c t i o n s.
T h e r e f o r e i t c a n b e u s e d t o s t o p e n z y m a t i c r e a c t i o n s f r o m o c c u r r i n g, s i n c e t h e y u s u a l l y r e q u i r e
t h e s e c a t i o n s.
T o m a k e 1 l i t e r:
!1 ] W e i g h o u t 1 4 6.1 3 g o f E D T A,
!2 ] A d d 8 0 0 m L d d w a t e r.
!3 ] S t i r v i g o r o u s l y a n d t h e n a d d a p p r o x i m a t e l y 2 0 g ( n o t m o r e ) o f s o d i u m h y d r o x i d e
! ! p e l l e t s.
!4 ] S t i r f o r a f u r t h e r 1 0 m i n s.
!5 ] A d j u s t t h e p H t o 8.0 w i t h m o r e p e l l e t s o r u s i n g a 1 0 N s o d i u m h y d r o x i d e s o l u t i o n.
! T h e E D T A w i l l n o t g o i n t o s o l u t i o n u n t i l a l m o s t p H 8.
!6 ] O n c e t h e E D T A s o l u t i o n h a s b e c o m e c l e a r c h e c k t h e p H o n c e m o r e.
!7 ] A d j u s t t h e v o l u m e t o 1 l i t e r.
!8 ] D i s p e n s e i n t o 1 0 0 m L b o t t l e s a n d a u t o c l a v e o n l i q u i d c y c l e f o r 2 0 m i n.
Q u a n t i t y n e e d e d:
!!I t m a y b e s u f fi c i e n t t o m a k e 5 0 0 m L a t a t i m e, d e p e n d e n t o n d e m a n d.
1M

G
LUCOSE
Glucose is also know as Dextrose, and D-Glucose.
!To make 500 mL
!!!
!!!A d d 9 0.0 8 g o f A n h y d r o u s D e x t r o s e
!!!D i s s o l v e i n d H
2
O
!!!Dilute to final volume with dH
2
O
!!!Filter Sterilize
IPTG

S
TOCK
S
OLUTION
(0.12
G
/
M
L)
IPTG is used to induce RNA production from the lac operator bacterial promoter
!!!!
2 5 m L !!!5 0 m L
!I P T G!!!3 g!!!6 g
!!D i s s o l v e i n w a t e r a n d b r i n g t o v o l u m e
!!F i l t e r s t e r i l i z e
!!A l i q u o t 1 m L i n t o u n s i l a n i z e d e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o
!!!d i s t i n g u i s h f r o m o l d e r b a t c h i f p o s s i b l e.
!!L a b e l w i t h a n ‘ I ’ o n t h e l i d.
LB

M
EDIA
LB is a nutrient rich media used to grow
E. coli
. We generally use LB to grow bacteria which
contain a plasmid so we can make large quantities of DNA stocks for other applications.
!!!!!!
1 L!!5 0 0 m L
!T r y p t o n e!!!!1 0 g!!5 g
!Y e a s t E x t r a c t!!!!5 g!!2.5 g
!N a C l!!!!!5 g!!2.5 g
A u t o c l a v e
A d d 1 µ L / 1 m L A M P ( 1 0 0 m g/ m L )
LB

M
EDIA
P
LATES
LB is a nutrient rich media used to grow
E. coli
. We generally use LB to grow bacteria which
contain a plasmid so we can make large quantities of DNA stocks for other applications.
!!!!!!
1 L!!5 0 0 m L
!T r y p t o n e!!!!1 0 g!!5 g
!Y e a s t E x t r a c t!!!!5 g!!2.5 g
!N a C l!!!!!5 g!!2.5 g
!B a c t o - A g a r!!!!2 0 g!!1 0 g
A u t o c l a v e
A d d 2 5 µ L A M P ( 1 0 0 m g/ m L ) / p l a t e
M9
MEDIA
M9 media is used for growing E. coli cultures. M9 media minus casamino acids is used to grow
E. coli when we want to label newly synthesized proteins with radiolabel so we can monitor
them. The radiolabel we commonly use is the amino acid methionine (
35
met) therefore we don't
want any non-radiolabelled (or cold) methionine (which is a constituent of casamino acids)
present.
For 1 liter: !100 mL 10x M9 salts
!!1 mL 1M CaCl
2
!!1 mL 0.1 M magnesium sulfate
!Add dd water to 850 mL.
!!Dissolve 8g glucose in 50 mL of dd water then add this dropwise, stirring
!!!vigorously.
!Increase the volume to 950 mL and autoclave.
!!Add 50 mL of 200g /liter casamino acids after autoclaving (for
M9 media minus
!!!casamino acids
omit this step and increase the volume to 1 liter before
!!!!autoclaving).
M9

M
INIMAL
M
EDIA
P
LATES
!For 500 ml:
!!
A d d 1 0 g D i f c o A g a r t o 4 2 5 m l d d H
2
0
!A u t o c l a v e 1 2 m i n.
!C o o l 1 5 m i n i n w a t e r b a t h.
!!A d d 5 0 m l 1 0 x M 9 s a l t s
!!A d d 5 m l 4 0 % G l u c o s e
!A d d r e q u e s t e d a d d i t i v e s:
!+ A m p!!3 0 0 u l f o r 5 0 0 m l
!- L !!2.5 m l T r p, 1 0 m l U r a
!- U!!2.5 m l T r p, 2.5 m l L e u
!- T!!2.5 m l L e u, 1 0 m l U r a
!S t i r o n s t i r p l a t e.
!P o u r p l a t e s a s u s u a l ( 5 0 0 m l m a k e s 1 s l e e v e ).
M9

P
LATES
M9 media is used for growing E. coli cultures. M9 media minus casamino acids is used to grow
E. coli when we want to label newly synthesized proteins with radiolabel so we can monitor
them. The radiolabel we commonly use is the amino acid methionine (
35
met) therefore we don't
want any non-radiolabelled (or cold) methionine (which is a constituent of casamino acids)
present.
For 1 liter: !100 mL 10x M9 salts
!!50 mL of 200g /liter casamino acids *
!!8 g glucose #
!!1 mL 1M CaCl
2
!!1 mL 0.1 M magnesium sulfate
!!20 g Agar
!Add dd water to 850 mL.
!# Dissolve glucose in 50 mL of dd water then add this dropwise after the volume has
!been increased to 850 mL, stirring vigorously.
!Increase the volume to 950 mL and autoclave.
!* Add this after autoclaving, for M9 media minus casamino acids omit this step and
!increase the volume to 1 liter before autoclaving.
10
X
M9
SALTS
M9 salts are a constituent of M9 media: see page 13
For 1 liter: !60g Na
2
HPO
4
anhydrous (Sodium Phosphate anhydrous, MW: 141.96)
!!30g KH
2
PO
4
anhydrous (Potassium Phosphate monobasic, MW: 136.09)
!!5g NaCl (sodium chloride, MW: 58.44 )
!!10g NH
4
Cl (ammonium chloride, MW: 53.49)
!Dissolve in 900 mL of dd water and then adjust the volume to 1 liter.
!Autoclave on
liquid cycle
for 20 mins.
1M
MAGNESIUM CHLORIDE
(MW 203.3g)
!For 1 liter:
!!Dissolve 203.3g of MgCl
2
•6H
2
0 in 800 mL of dd water.
!!Adjust volume to 1 liter.
!!Dispense into 100 mL bottles
!!Autoclave on
liquid cycle
for 20 mins.
!!
!Q u a n t i t y n e e d e d:
!!I t m a y b e s u f fi c i e n t t o m a k e 5 0 0 m L a t a t i m e d e p e n d e n t o n d e m a n d.
1M

M
G
C
L
2

(M
AGNESIUM CHLORIDE
),

1M

M
G
SO
4

(M
AGNESIUM
SULFATE
)
For 500 mL: !101.65g MgCl
2
•6H
2
0 (MW 203.30)
!!123.23g MgS0
4
•7H
2
0 (MW 246.47)
!Should be dissolved in 450 mL dd water.
!The volume should then be adjusted to 500 mL,
!dispensed in 100 mL aliquots
!Autoclaved for 20 min. on
liquid cycle
.
N
EUTRALIZATION
S
OLUTION
!For 500 ml:
!!147.2 g Potassium Acetate
!!57.5 ml Glacial Acetic Acid
No sterilization required.
10
X
MMR
To make 1 litre of 10 x MMR:
!Dissolve!58.44g sodium chloride (MW: 58.44g)
!!!1.49g pottasium chloride (MW: 74.56g)
!!!11.92g HEPES (MW: 238.31g )
!!!in 900 ml of dd water. Adjust the pH to 7.4 using 10M NaOH. Incresae
!!!!the volume to 1 litre and filter sterilise.
This solution is only to be made on demand.
We use 1 x MMR (100 mM NaCL, 2 mM KCl, 5 mM HEPES) to bathe our frog oocytes in once
they are removed from the frog. Oocytes can be used for micro-injection experiments or matured
(to form an egg which is ready to undergo fertilisation) for about 24 hours after surgery when
they are maintained in this buffer.
20
X
MOPS
For 1 Liter:
!Dissolve in 500 ml H
2
O
!!
!!8 3.7 2 g M O P S
!!8.2 0 g S o d i u m A c e t a t e
!!7.4 4 g E D T A
!A d j u s t p H t o 7.0 w i t h 1 0 M N a O H
!A d j u s t t o 1 L
!A u t o c l a v e ( w i l l t u r n y e l l o w a f t e r b e i n g a u t o c l a v e d ).
P
E N
-S
TREP
1000

X

S
TOCK
S
OLUTION
Pen-Strep = Penicillin-Streptomycin. These two antibiotics are used in our buffers we store frog
oocytes in. The antibiotic keeps fungi, and bacteria from growing on the oocytes, thus keeping
the oocytes relatively healthy.
!!To make add the following to dH
2
O
!!!!10 mg/mL penicillin
!!!!10 mg/mL streptomycin
!Filter sterilize and store in 1 mL aliquots at -20˚C. in unsilanized eppendorf
!!!!tubes. Use a different color to distinguish from older batch if
possible.
!Label with an ‘S’ on the lid.
P
HOSPHATE
B
UFFER
!
Part 1:
!!Place 2160 ml ddH
2
O in a 4 L Flask
!!Add 340.23 g KH
2
PO
4
!
Part 2:
!!
!!P l a c e 6 2 0 m l d d H
2
O i n a 4 L B e a k e r
!!A d d 1 3 0.6 4 K
2
P O
4
!
P a r t 3:
!!A d d S o l u t i o n 1 t o S o l u t i o n 2 u n t i l p H 6 i s r e a c h e d.
!!S t o r e i n c a r b o y
!!D i s p e n s e i n t o 7 5 m l b o t t l e s f o r N G M p o u r i n g.
M a k e o n l y o n d e m a n d!!
1
X
PBS

(P
HOSPHATE
-B
UFFERED
S
ALINE
)
!Dissolve in 800 ml ddH
2
0:
!!8.0 g NaCl
!!0.2 g KCl
!!1.44 g Na
2
HPO
4
!!0.24 g KH
2
PO
4
!pH to 7.4 with HCl or NaOH
!adjust volume to 1L with water and autoclave in 500 ml bottles.
10
X
PBS
!Dissolve in 800 ml ddH
2
O:
!!80 g NaCl
!!2.0 g KCl
!!14.4 g Na
2
HPO
4
!!2.4 g KH
2
PO
4
!pH to 7.4 with HCl or NaOH
!Adjust volume to 1 L with water and autoclave in 500 ml bottles.
P
IPET
C
LEANING
P
ROTOCOL
Glass pipets need to be cleaned very carefully to ensure that they are exceptionally free of all
contaminants.
Dirty pipets are placed in bleach baths around the lab. They need to be emptied two - three times
a week, pipets cleaned, and the water changed (water only needs to be changed if cloudy).
!Protocol for Pipet Baths:
!!!1] Fill each bath 3 inches above the top of pipets
!!!2] Put approximately 2 cap fulls of bleach in each bath
!!!
To clean pipets we soak them in Sulfuric Acid with Nochromix added. The acid bath ensures that
all contaminants are removed from the pipets, this includes, RNases.
!Protocol for Acid Bath
!You mus
t
wear your lab coat, thick rubber gloves, and safety glasses when washing
!pipets after this point. Sulfuric acid will burn your cloths as well as your skin!!!!
!!1] Add 1 packet of Nochromix to an entire jug (2.5 L) of Sulfuric Acid (H
2
SO
4
)
!!2] Pour the Sulfuric Acid/Nochromix solution into the acid bath until full
!!Make sure acid covers the pipets completely.
!Washing Protocol:
!!1] Remove pipets from bleach baths and put into the washing container.
!!!!Separate the 25 ml and 10 ml pipets into separate batches. Place
25 ml !!!!tip down and 10 ml tip up.
!!2] Rinse with distilled water in pipet washer in undergrad room for 15 mins
!!3] Drain
!!4] Lift out and put in acid bath in dish washing room down by dishwasher.
!!5] Soak in acid bath overnight. Flip 25 ml Pipets tip up so they drain well and
!!!!soak over night again.
!!6] Drain well and be careful not to drip acid around the sink
!!7] Rinse any spilled acid down the drain.
!!8] Rinse pipets in the pipet cleaners in dish washing room by setting timers at
!!!1 hour with tap H
2
O
!!!then
!!!1 hour with dH
2
O
!!Only set timers, do not touch faucet
!!9] Put pipets into drying oven for 40 mins at setting 3.
!Warning: Do not use a higher setting, as it will melt the plastic pipet holders
!!
1 0 ] S e p a r a t e p i p e t s i n t o 1, 1 0, a n d 2 5 m L a n d a l s o i n b l o w o u t v s. n o n - b l o w o u t
!!1 1 ] P u t i n t o m e t a l c a n i s t e r s
!!1 2 ] B a k e i n o v e n a t 4 0 0 ˚ F f o r 4 h o u r s.
1

M
POTASSIUM CHLORIDE
(MW 74.56)
!For 1 liter:
!!Dissolve 74.56g of KCl in 800 mL of dd water.
!!Adjust volume to 1 liter.
!!Dispense into 100 mL bottles
!!Autoclave on
liquid cycle
for 20 mins.
!Quantity needed:
!
!It may be sufficient to make 500 mL at a time dependent on demand.
P
ROTEINASE
K

(10
MG
/
M
L)
Proteinase K is an enzyme which degrades proteins, and can be used to help separate DNA/
RNA from proteins when purifying nucleic acids.
!!!!!!
1 0 m L
!P r o t e i n a s e K!!!!1 0 0 m g
!!D i s s o l v e i n w a t e r a n d b r i n g t o v o l u m e
!!A l i q u o t 1 m L i n t o e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o d i s t i n g u i s h f r o m
!!!!o l d e r b a t c h i f p o s s i b l e. L a b e l w i t h a n ‘ K ’ o n t h e l i d.
!!I n c u b a t e a t 3 7 ˚ C f o r 1 h o u r
!!!I n c u b a t i o n a l l o w s p r o t e i n a s e K t o d i g e s t a n y n u c l e a s e s w h i c h m i g h t b e
!!!i n t h e s o l u t i o n.
!!S t o r e a t - 2 0 ˚ C
RN
ASE
A

S
TOCK
S
OLUTION
(10
MG
/
M
L)
RNase A is normally the enemy in this lab (among many other RNases), however, many times we
actually want to degrade RNA, such as when we wish to separate RNA from DNA, and then
purify DNA. So we do need to have this around. You should always be conscious, however, that
when you make RNaseA you are exposing the glassware and everything else you may touch
while making RNaseA to an incredible concentration of RNases. It would be wise and
appreciated if you made sure that you change your gloves after making RNaseA, so you don't
spread it around the lab as you work.
!!
!!!!!!
2 0 m L!!!5 0 m L
!! R N a s e A !!!2 0 0 m g ( 0.2 g )!!5 0 0 m g ( 0.5 g )
!! 1 M T r i s p H 7.5!!!2 0 0 µ L!!!5 0 0 µ L
!! N a C l !!!!6 0 µ L!!!1 5 0 µ L
!!M a k e 1.0 m L a l i q u o t s i n u n s i l a n i z e d e p p e n d o r f t u b e s. U s e a d i f f e r e n t c o l o r t o
!!!!d i s t i n g u i s h f r o m o l d e r b a t c h i f p o s s i b l e. L a b e l w i t h a n ‘ R N ’ o n
t h e l i d.
!!H e a t t o 1 0 0 ˚ C i n w a t e r b a t h f o r 1 5 m i n s ( k i l l s a n y c o n t a m i n a t i n g D N a s e s )
!!C o o l t o r o o m t e m p e r a t u r e
!!S t o r e i n - 2 0 ˚ C f r e e z e r i n h o t r o o m
S
ILANIZED
G
LASS
S
LIDES
!When we do micro-injections of materials into frog oocytes or eggs we draw very small
volumes of concentrated materials up into the injection needle. To make it slightly easier to load
the needle we normally silanise the glass slides onto which we pipette the material to be loaded.
The silanising makes the drop of material bead nicely on the slide ie. it forms a concentrated
drop rather than spreading out on the slide.
Protocol:

!1. Dip slides into a large weighing boat containing 5% dimethly dichloro silane and
!95% chloroform (bottle in the fume hood). This procedure should be performed in the
!fume hood to avoid fume inhalation. The slides should be dipped using forceps.
2. Dry the slide briefly by placing on a kimwipe. Make sure there are no spots, wipe
slides with a kimwipe. If spots will not come off, wipe with ethanol on both sides until
dry. Wrap two slides together in aluminium foil.
!3. Place in a pyrex dish and bake at 400 degrees for 4 hours.
Quantity needed:
Silanise a box at a time.
Caution:
Work in the fume hood and wear gloves.
10%

SDS

(
SODIUM DODECYL SULFATE
)
(MW: 288.38)
SDS is a detergent which has many uses in molecular biology, such as lysing cells and
denaturing protein
!Percentage solutions are made weight/volume. i.e. a 10% solution contains 10g in 100
!mL.
!!W e i g h 5 0 g a n d d i s s o l v e i n 4 5 0 m L d d w a t e r.
!!I t w i l l b e n e c e s s a r y t o h e a t t h e s o l u t i o n t o 6 8 d e g r e e s t o a s s i s t d i s s o l u t i o n.
!!A d j u s t t h e v o l u m e t o 5 0 0 m L.
!!F i l t e r s t e r i l i z e, i n 1 0 0 m L u n i t s,
d o n o t a u t o c l a v e.
!W e a r a m a s k w h e n w e i g h i n g S D S i t i s h a r m f u l e s p e c i a l l y w h e n i n h a l e d. A l a b c o a t
!a n d s a f e t y g l a s s e s w i l l n o t h u r t e i t h e r.

SDS-P
AGE
R
UNNING
B
UFFER
(10X)
!For 1 L:
!!30.3 g Tris Base
!!144.1 g Glycine
!!10.0 g SDS (Electro grade)
!pH to 8.3 with HCl
!Filter sterilize
SDS-P
AGE
S
EPARATING
B
UFFER
(4
X
)
This is u sed in combination with SDS-Page Stacking buffer for running gels and separating
proteins.
For 250 mL
Dissolve in 250 mL water
!Tris base!!!45.0g
!SDS (electrophoresis grade)!1.0g
Adjust pH to 8.8 with Hcl (about 6 mL).
Filter sterilize and store at 4°C.
Quantity: 1
SDS-P
AGE
S
TACKING
B
UFFER
(4
X
)
Used for running gels and separating proteins.
For 250mL
Dissolve in 250 mL water
!Tris base !!!15.12g
!SDA (electrophoresis grade)!1.0g
Adjust pH to 6.8 with HCl.
Filter sterilize and store at 4 C.
Quantity: 1
SDS-P
AGE
S
TAIN
/D
ESTAIN
!Stain
!!Create a solution that is:
!!!0.25% Coomaissie Blue
!!!50.0% MeOH
!!!10.0% HOAc
!Destain
!!Create a solution that is:
!!!40.0% MeOH
!!!10.0% HOAc
SDS

G
EL
L
OADING
D
YE
(2X)
!For 10 ml:
!!2.5 ml Stacking buffer (4X)
!!2.0 ml Glycerol
!!0.5 ml Bromophenol Blue (0.5%)
!!0.6 g SDS (electro grade)
20
X
SSC

(S
ODIUM
C
HLORIDE
/

S
ODIUM
C
ITRATE
)
For 8 Liters:!1402.4 g NaCl
!!705.6 g Sodium Citrate
!Dissolve in 8 L of ddH
2
O
!No need to autoclave
!May take time to get into solution
3M
SODIUM ACETATE P
H

5.2

(MW 82.03g)
!For 500 mL:
!!Dissolve 123.05g of sodium acetate in 400 mL of dd water.
!!Adjust pH to 5.2 with glacial acetic acid.
!!Adjust volume to 500 mL.
!!Dispense into 100 mL bottles
!!Autoclave on
liquid cycle
for twenty minutes.
!Quantity needed:
!!
!!
I t m a y b e s u f fi c i e n t t o m a k e 5 0 0 m L a t a t i m e d e p e n d e n t o n d e m a n d.