Please find the link to the following protocol

In Vitro Disintegration Assay for P Element Transposase


Preparation of the Y Oligomer: Kinasing Oligonucleotides, Annealing and Gel Purification

1. The standard substrate oligonucleotides are T1 (16-mer), T3
(30-mer), P1/P2L (84-mer), and P2L (70-mer). The T1
oligonucleotide is labeled by phosphorylation with
γ-[32P]-ATP and T4 polynucleotide kinase and then annealed
to P1/P2L, P2L and T3.
Then add 60 μl TEMED.

9. Add 40 μl of dye-sucrose mix to the Y oligomer sample and
run the sample at 200 V until the Bromophenol Blue dye has
migrated about two-thirds the way down the gel.

10. Autoradiograph and excise the slow-moving Y oligomer. The
unlabeled T1 will migrate faster, as will the free γ-[32P]-ATP.

11. Excise the band and put it in a 1.5 ml microcentrifuge tube.
Elute the DNA in 0.5 M Ammonium Acetate/10 mM
Magnesium Acetate. Incubate the elution/gel mixture for 12
hours at 4°C on a shaking platform. Remove the liquid and
repeat the elution 3 more times. Store each elution at 4°C.

12. Pool the four elution volumes in an Amicon Centricon-10 and
store at 4°C during the other elutions.

13. Centrifuge the Centricon-10 at 9,700 X g (6,000 rpm using a
Sorvall SS34 rotor) at 4°C to concentrate the sample.

14. Add 2 ml of ice-cold TEN to the concentrated sample and
reconcentrate. Add an additional 2 ml of ice-cold TEN to the
sample and reconcentrate to a final volume of 50-100 μl.

15. Invert the Centricon-10, centrifuge for 1 min at 9,700 X g
(6,000 rpm using a Sorvall SS34 rotor) at 4°C and store the
retentate at -20°C.



Disintegration Assay (both GTP and MgCl2 are essential cofactors for this reaction)

1. Use 2 μl of the Y oligomer per assay.

2. Set up the following reaction in 15 μl total volume: 2 μl Y
oligomer, 1.5 μl 10 mM GTP, 1.5 μl 100 mM manganese
chloride, 7 μl HGKED, 3 μl protein fraction of interest (in 0.1M
KOH)

3. Incubate at 25°C or 30°C for 1 hr.

4. Add 15 μl deionized formamide dyes/30 mM EDTA.

5. Boil the samples and load 4 μl on a 15% urea polyacrylamide
0.4 mm thick sequencing gel using a clear 24-tooth comb.
The Bromophenol Blue co-migrates with 10 nucleotide
oligonucleotides on this percent gel. For a 40 ml solution to
prepare a 15 % polyacrylamide urea gel, combine: 20 g
Urea, 15 ml 40 % Acrylamide (19:1), 8.2 ml ddH2O, 0.4 ml
10 % Ammonium Persulfate (filter this mixture through a
Whatman #1 filter), add 25 μl TEMED.

6. Transfer the wet gel to an film cassette and cover it with
plastic wrap. Expose the gel to X-ray film at -80°C with an
intensifying screen.

Solutions:

5 M NaCl
P2L.............................505 μg/ml of P2L Oligonucleotide Primer
T3................................980 μg/ml of T3 Oligonucleotide Primer
P1P2L......................270 μg/ml of P1P2L Oligonucleotide Primer
Oligo Kinase Buffer (10X)......0.1M MgCl2
......0.15M DTT
......0.66M Tris-HCl, pH 7.6
100 mM MnCl2...............................................Do not autoclave
10 mM GTP
30% (w/v) acrylamide...............................CAUTION Biohazard
(30:0.8 acrylamide:bisacrylamide)
40% (w/v) acrylamide...............................CAUTION Biohazard
(19:1 acrylamide:bisacrylamide)
0.25 M EDTA
HGKED.............................................Add DTT fresh before use
..............20 mM HEPES, pH 7.6 with potassium hydroxide
.................................................................0.5 mM DTT
...............................................................0.5 mM EDTA
.......................................................20% (v/v) Glycerol
..................................Add KOH to desired concentration
TEN....................................................................100 mM NaCl
......................................................................1 mM EDTA
.....................................................10 mM Tris-HCl, pH 7.5
10 mM Magnesium Acetate
0.5 M Ammonium Acetate
1 M MgCl2.....................................................Do not autoclave
Sucrose Dye Buffer (10X).......Add a pinch of Bromophenol Blue
...............70% Sucrose (w/v) 1 X TBE
10% (w/v) Ammonium Persulfate
TBE buffer (20X)........................................0.4 M EDTA, pH 8.0
.....................................................1.8 M Tris
............................................1.8 M Boric Acid



Links and References:

http://www.bio.com/protocolstools/protocol.jhtml?id=p497

Chow SA, Vincent KA, Ellison V, Brown PO. Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science 1992; 255:723-6