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Creation and use of your infectious vector

Stewart Laboratory Standard Operating Procedure               


TRAP Assay

A. Prepare lysate

Lyse cells for 20-30 on ice in CHAPS lysis buffer.

Spin cells at 4C for 20

Save supernatant in a new Rnase-free tube.

Quantitate protein concentration using the Lowry protocol. Be sure to have appropriate blanks and concentration controls.

Dilute a small portion of the sample to 50 ng/μl. Return the undiluted sample to -70C for future use.

Take one quarter of the diluted sample and boil for 5 and crash on ice. This will serve as your heat inactivated control.

B. End label TS oligo:

Label as follows (this makes enough oligo for 10 reactions):

2.5 μl g32P ATP (3000 Ci/mmol)

2 μl 10X buffer

5.2 μl TS primer

9.8 μl ddH2O

0.5 μl T4 kinase

o Heat to 37C for 20

o Heat to 85C for 5

C. Set-up TRAP reaction:

Set-up the following 50 μl reaction (this is per reaction so when performing multiply reactions prepare a supermix). Each sample should have a minimum of two conditions, the sample and the heat inactivated control. You can also set-up a dilution series if you are attempting to quantitate the activity:

5 μl 10X buffer

2 μl Primer

2 μl labeled TS primer

1 μl dNTPs (at 2.5 mM each)

0.4 μl Taq

37.6 μl ddH2O

2 μl prepared extract

D. Run reaction

Set up PCR machine as follows:

30 at 30C

27 cycles with the following:

94C for 30

60C for 30

E. Run gel:

12.5% acrylamide gel (0.4mm) with 0.5X TBE buffer

add 10 μl of 6X loading dye and run 6 μl

run gel at about 1000-1200 volts until the first dye runs off and the second dye is about 3/4 the way into the gel.

Important buffers:

1X CHAPS (3-[(Cholamidopropyl)dimethylammonio]-1-propanesulfonate) buffer (All Rnase-free)

10 mM Tris, pH 7.5

1 mM MgCl2

1 mM EGTA

0.1 mM benzamidine

5 mM 2-mercaptoethanol

0.5% CHAPS

10% glycerol

o Immediately before lysing cells add 5 mM -mercaptoethanol (stock from Sigma is at 14.3 M so add 0.5 μl to 1.4 mls of the buffer)

10X TRAP buffer

200 mM Tris, pH 8.3

15 mM MgCl2

630 mM KCl

0.5% Tween 20

10 mM EGTA

0.1% BSA

50X dNTPs

2.5 mM of each dNTP

Oligos:

ACX GCGCGGCTTACCCTTACCCTTACCCTAACC

TSNT AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT

NT ATCGCTTCTCGGCCTTTT

TS AATCCGTCGAGCAGAGTT

Oligos ordered purified in solution

Concentrations for use:

Primer mix II

ACX 0.1 μg/sample

NT 0.01 μg/sample

TSNT 0.01 amol/sample

Primer mix III

ACX 0.1 μg/sample

NT 0.1 ng/sample

TSNT 0.01 amol/sample