TOEPRINTING FOLLOWING TRANSLATION IN NEB

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TOEPRINTING FOLLOWING TRANSLATION IN NEB-PURExpress SYSTEM

(∆RIBOSOME/∆AA/∆tRNA kit)

5’ end labeling of primer with



32

P

MATERIALS:

Toeprinting/sequencing primer 20 pmoles/µL

- Label enough for sequencing (1.5 µL /rxn) and toeprint (0.5 µL/rxn)

γ-

32

P-ATP at 6000 Ci/mmol

10X Buffer A (Fermentas, comes with PNK enzyme)

T4-PNK (10U/ µL from Fermentas Cat No. EK0031)

PROTOCOL:

Set up reaction in PCR tube:

Reagent Volume (µL)

5.5 dH

2

O

10X Buffer A 1

Primer

γ-32P-ATP

1

1.5

T4-PNK

Total

1

10

Incubate 30 min at 37 o

C

Inactivate 2 min at 95 o

C

Keep on ice until use

Translation reactions

MATERIALS:

Template:

PCR fragment (must contain T7 promoter sequence) (0.1 to 0.3 pmoles/µL) or

T7 transcript (1-10 pmoles/µL)

RNAse inhibitor (40U/µL from Fermentas Cat No. EO0382)



32

P labeled primer

From the NEB PURExpress kit ∆RIBOSOME/∆AA/∆tRNA kit (a custom made combination of the E3313 and E6840 kits):

-Solutions A and factor mix (thawed on ice, quick spin, and kept on ice)

Ribosomes (20 pmoles/µL)

Antibiotics stock (if needed):

-can be directly added to the translation reaction if the stock is in water, or

-if there is no room for this component in the translation reaction, stock can be prepared in EtOH, added to the corresponding tube(s), dried in speed vac and resuspended directly with the translation mix

PROTOCOL:

Set up reactions in pre-chilled eppendorf tubes

Translation reaction (5 µL):

Component

Solution A (∆AA-

∆tRNA)

AA stock (3 mM) tRNAs

Factor mix

Template

Volume (µL)

1

0.5

0.5

0.6

X

γ-32P labeled primer 0.5

RNase inhibitor

Ribosomes antibiotic, other components, or dH2O

0.2

0.5 to 5

Vortex gently and briefly, do quick spin

Incubate at 37°C, 30 min (or optimize as needed)

Reverse transcription

MATERIALS:

Pure System Buffer (PSB)

Reverse transcriptase (Roche, Cat #10109118001 21 U/ µL) freshly diluted 1:5 in

PSB

4 mM dNTP solution (in water)

10 N NaOH

HCl (concentrated, 12N)

PROTOCOL:

Mix (right before use) diluted RT and dNTP solution 1:1

Add 1 µL of the mix/ translation reaction

Incubate at 37 C for 5-10 min

Add 1 µL NaOH solution to extension reactions, vortex gently

Incubate at 37 C for 15 min

Add 0.8 µL HCl

Samples preparation for sequencing gel

MATERIALS:

Resuspension buffer

Glycoblue (15 mg/mL, Ambion, Cat No. AM9515)

Phenol

Chloroform

EtOH (Absolute)

70% EtOH (cold)

Eppendorf centrifuge cooled to 4 C

PROTOCOL:

Add 200 µL of resuspension buffer

Extract 5 min with 180 µL volume of phenol

Extract 5 min with 180 µL volume chloroform

Add 600 µL volumes of cold ethanol

Add 0.5 µL of glycoblue

Incubate at least 10 min at -80 C

Spin at least 30 min in the cold centrifuge

Keep samples on ice

Discard supernatant (to radioactive container)

Wash pellets with cold 70% EtOH

Spin for at least 2 min in cold centrifuge

Discard supernatant (to radioactive container)

Quick spin, finish removing remaining supernatant

Air-dry pellets for 5 min at room temp or 1 min in speed vac

Resuspend pellets with 8 µL of formamide dye

Heat at 95 C for 2 min

Load 1.5 µL in 6% sequencing gel along with sequencing reactions

Solutions

Pure System Buffer

Pure System Buffer (PSB)

1

: 9 mM Mg-Acetate, 5 mM K-phosphate, pH 7.3, 95 mM Kglutamate, 5 mM NH

4

Cl, 0.5 mM CaCl

2

, 1 mM spermidine, 8 mM putrescine, 1 mM

DTT.

Stock Volume (mL) for 10 mL PSB

1 M Mg-Ac

0.5 M K-phosphate, pH 7.3

1 M K-glutamate

1 M NH

4

Cl

0.5 M CaCl

2

1 M spermidine

90

100

950

50

10

10

0.1 M putrescine

0.1 M DTT

800

100

Bring to 10 mL with water

Filter

Keep at – 20 o

C in ~1 mL aliquots

1

Shimizu, Y., A. Inoue, Y. Tomar, T. Suzuki, T. Yokogawa, K. Nishikawa, and T. Ueda.

2001. Nature Biotechnology. 19: 751-755

Resuspension buffer

0.3M Na-o-Ac, 5 mM EDTA, 0.5% SDS

3M Na-o-Ac (pH 5.5)

0.5M EDTA (pH 8)

10% SDS

Bring to 10 mL with water

Store at room temperature

1 mL

Sequencing Reactions based on the former fmol sequencing protocol from Promega (now discontinued)

MATERIALS:

5X fmol Sequencing Buffer (250 mM Tris-HCl, pH 9.0, at 25 C, 10 mM MgCl

2

)

AmpliTaq DNA Polymerase (Stoffel Fragment, Applied Biosystems Cat. No. N808-

0038, 10 U/µL)

IMPORTANT:

For sequencing reactions AmpliTaq Polymerase is diluted 1:5 in storage buffer.

Diluted enzyme can be kept at -20C for few months with no noticeable loss of activity.

Terminator solutions G, A, T, C

Template (150 ng/µL)

PROTOCOL:

Mix in eppendorf tube

Component Volume (µL) dH

2

O

5X buffer

γ-32P labeled primer 1.5

Template diluted AmpliTaq

Total

Vortex gently, quick spin

8.5

5

1

1

17

Distribute 4 µl of the mix into PCR tubes already containing 2 µL of terminators G, A,

T, C

Vortex gently, quick spin

PCR program:

95C, 2 min then, 30 cycles of

95C, 30 s

42C, 30 s

70 C, 1 min

Add 3 µL of formamide dye

(reactions can be stored at -20 C or used immediately)

Heat for 2 min at 95 C right before running in sequencing gel

Load 1.5 µL in sequencing gel

Solutions

5X fmol Sequencing Buffer

250 mM Tris-HCl, pH 9.0, at 25 C

10 mM MgCl

2

)

Storage Buffer for Amplitaq DNA Polymerase, Stoffel Fragment:

100 mM KCl

20 mM Tris-HCl, pH 8.0 (at room temp)

0.1

mM EDTA

1 mM DTT

50% (v/v) Glycerol

0.5% (w/v) Nonidet P40 (Igepal Ca-630, Sigma, Cat. No. I-8896)

0.5% (w/v) Tween 20 (Sigma Cat. No. P1379-500 mL)

Terminators:

dNTP set (Roche Cat. No. 11969064001) ddNTP set (Roche Cat. No. 03732738001)

7-Deaza-dGTP, Li-salt (Roche Cat. No. 10988537001)

Nucleotide mixes

Component Deaza G Deaza A Deaza T ddGTP (10mM) ddATP (10 mM) ddTTP (10 mM) ddCTP (10 mM)

7-deaza dGTP (10 mM)

30 µM

0.9 µl -

- 350µM

-

10.5µl -

Deaza C

-

-

- - 600 µM  18 µl -

- - -

20 µM

0.6 µl 20 µM

0.6 µl 20 µM

0.6 µl

200 µM  6 µl

20 µM

0.6 µl dATP (10 mM) dTTP (10 mM) dCTP (10 mM)

20 µM

0.6 µl 20 µM

0.6 µl 20 µM

0.6 µl

20 µM

0.6 µl 20 µM

0.6 µl 20 µM

0.6 µl

20 µM

0.6 µl 20 µM

0.6 µl 20 µM

0.6 µl

20 µM

0.6 µl

20 µM

0.6 µl

20 µM

0.6 µl

Water 296.7 µl 287.1 µl 279.6 µl

300 µl Total

Sequencing Stop Solution

9.8 ml formamide (Formamide, deionized, nuclease free, Ambion, cat# 9342)

200 µl 0.5 M EDTA pH 8.0

10 mg bromophenol blue

10 mg xylene cyanol

291.6 µl

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