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 32P
MATERIALS:
Toeprinting/sequencing primer 20 pmoles/µL
- Label enough for sequencing (1.5 µL /rxn) and toeprint (0.5 µL/rxn)
γ-32P-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)
dH2O
5.5
10X Buffer A 1
Primer
1
γ-32P-ATP
1.5
T4-PNK
1
Total
10
Incubate 30 min at 37oC
Inactivate 2 min at 95oC
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)
32P 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
γ-32P labeled primer
RNase inhibitor
Ribosomes
antibiotic, other
components, or
dH2O
Volume (µL)
1
0.5
0.5
0.6
X
0.5
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 NH4Cl, 0.5 mM CaCl2, 1 mM spermidine, 8 mM putrescine, 1 mM
DTT.
Stock
Volume (mL) for 10 mL PSB
1 M Mg-Ac
90
0.5 M K-phosphate, pH 7.3
100
1 M K-glutamate
950
1 M NH4Cl
50
0.5 M CaCl2
10
1 M spermidine
10
0.1 M putrescine
800
0.1 M DTT
100
Bring to 10 mL with water
Filter
Keep at – 20oC in ~1 mL aliquots
Shimizu, Y., A. Inoue, Y. Tomar, T. Suzuki, T. Yokogawa, K. Nishikawa, and T. Ueda.
2001. Nature Biotechnology. 19: 751-755
1
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 MgCl2)
AmpliTaq DNA Polymerase (Stoffel Fragment, Applied Biosystems Cat. No. N8080038, 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
dH2O
5X buffer
γ-32P labeled primer
Template
diluted AmpliTaq
Total
Volume (µL)
8.5
5
1.5
1
1
17
Vortex gently, quick spin
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 MgCl2)
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)
Component
Deaza G
Nucleotide mixes
Deaza A
Deaza T
Deaza C
ddGTP (10mM)
ddATP (10 mM)
ddTTP (10 mM)
ddCTP (10 mM)
7-deaza dGTP (10
mM)
dATP (10 mM)
dTTP (10 mM)
dCTP (10 mM)
30 µM 0.9 µl
20 µM 0.6 µl
350µM10.5µl
20 µM 0.6 µl
600 µM 18 µl
20 µM 0.6 µl
200 µM 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
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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