| Hybridization
of Overgos to BAC filters |
 |
This original protocol was developed by Dr John D. Mc Pherson
(Washington University School of Medicine, Genome Sequencing
Center)
Reagents
Hybridization solution
1 mM EDTA
7% SDS
0.5M sodium phosphate (pH7.2)
1M sodium phosphate, pH 7.2:
426 g sodium phosphate dibasic, anhydrous (Na2HPO4) in 1700
ml H2O, add 12 ml 85% H3PO4 and make to 3 liter
0.5M EDTA, pH 8.0:
200 g EDTA - 4Na and 176.44 g EDTA - 2Na in 1,600 ml H2O,
pH to 8.0 with NaOH pellet and make to 2 liter.
To make 4000 ml: (use sterile ddH2O)
1. To 2000 ml 1M sodium phosphate, add 1200 ml H2O, 8ml 0.5M
EDTA and 280 g SDS.
2. Heat and stir until the SDS is dissolved (1 hr or so).
3. Bring volume to 4000 ml.
4. Warm to 60 C prior to use.
Washing Buffer B:
1mM EDTA, 1 % SDS, 40mM Na2HPO4
12 ml 0.5 M EDTA
60 g SDS
240 ml 1M Na2HPO4, pH7.2
to 6L with ddH2O
Washing solution 2:
1.5x SSC, 0.1% SDS
375 ml 20x SSC
25 ml 20% SDS
to 5L with ddH2O
Washing solution 3:
0.5x SSC, 0.1% SDS
125 ml 20x SSC
25 ml 20% SDS
to 5L with ddH2O
20x SSC:
701.2 g NaCl
352.8 g sodium citrate, dihydrate
pH to 7.0 with a few drop of a conc. HCl
Bring volume to 4L with ddH2O
20% SDS: 400g SDS (Ultra Pure: Life Technologies)/2L
ddH2O
BSA (2 mg/ml) : diluted 10ml/ml BSA (purchased
from New England Biolabs) with ddH2O prior to use
Overgo labeling buffer: OLB {-A, -C}
Solution O: 1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2
625 ul 2 M Tris-HCl (pH8.0)
125 ul 1 M MgCl2
250 ul ddH2O
Solution A: 1 ml solution O
18 ul 2-mercaptoethanol
5 ul 100 mM dTTP
5 ul 100 mM dGTP
Solution B: 2 M HEPES-NaOH, pH 6.6
Dissolve 23.8g of HEPES-free acid (m.w. 238.3; SIGMA H-4034)
in 40ml de-ionized distilled water and adjust the pH to 6.6
with NaOH. Adjust the final volume to 50 ml with deionized
distilled water.
Solution C: 3 mM Tris-HCl, pH 7.4, 0.2mM EDTA
15 ul 1 M Tris-HCl (pH7.4)
2 ul 0.5 M EDTA
to 5 ml with ddH2O
Prepare OLB: A:B:C, 1:2.5:1.5
Solution A 1ml
Solution B 2.5ml
Solution C 1.5ml
Aliquot and store at -20 C.
Overgo Labeling Reaction: 10
ul reaction
1) Combine 0.5 ul of complementary 20 uM oligos (0.5 + 0.5)
with 4.5 ul ddH2O.
{10 pmol each oligo per reaction}
2) Heat solution of mixed oligos at 80 C, 5 min., 37 C, 10
min. Store on ice.
3) Add to the oligo mix:
BSA (2 mg/ml): 0.5 ul
OLB {-A, -C} 2.0 ul
32P-dATP* 0.5
ul *3000
Ci/mmol, 10 mCi/ml
32P-dCTP* 0.5
ul
Klenow fragment 1 ul (2U/ul)
4) Incubate at room temperature for 1 hour.
5) Remove unincorporated nucleotides with Sephadex G50 columns.
Hybridization of Overgos to
BAC filters
1) Filters are stacked 4-6 high, interleaved with nylon spacers
of equal size, in a plastic tray soaked with hybridization
solution. Press out air bubbles and loosely roll the filters.
Insert the rolled filters into a hybridization bottle the
same way for all bottles. Remove air bubbles from the filters
using a 25 ml glass pipette, making sure filters are flat.
Add 25 ml of warmed hybridization solution to each bottle.
Make sure that all filters are rolled in the same direction
and that the bottle is correctly placed in the oven to keep
them rolled. The rotation speed is set to 6.
2) Filters are pre-hybridized for 1 hour. Replace hybridization
solution before adding probes if filter is used for the first
time.
3) After filter pre-hybridization, heat the labeled probes
at 95 C on hot block for 10 min. Then immediately place probes
on slushy ice and add to appropriate hybridization bottle.
Probes are allowed to hybridize with filters overnight; however,
2-day hybridizations give somewhat stronger signals, especially
with older filters.
Washing: Preheat all wash solutions to 60 C
1) Hybridization solution is removed and the bottle filled
with 100 ml 1x washing buffer B. The bottle is returned to
the oven at 60 C and rotated for 30 minutes, speed 8.
2) Filters are then washed as follows in hybridization oven,
speed 8: (100 ml wash).
2x Washing solution 2 at 60 C for 20min
3) Filters are washed in the hybridization oven for 20 minutes
at 60 C with 100 ml wash solution 3, rotating speed 8.
4) Soak filters in hybridization solution.
Phospho-Imaging:
1) Place filters in plastic bags and remove all air bubbles.
Seal and check for leaks. Wipe outside of bag with wet kimwipe
to remove any hybridization solution.
2) Place filters in a phosphoimage cassette for overnight
exposure. Scan image into Storm Phosphoimager using: template
= filter, pixel = 200 um. (8 minutes to scan image). See Figure
1 for an example.
3) After image capture, filters are stored at -20 C in the
sealed plastic bag or in hybridization buffer in a plastic
bag at room temperature for a long-term storage.
References:
McPherson
JD, Marra M, Hillier L, Waterston RH, Chinwalla A, Wallis
J, Sekhon M, Wylie K, Mardis ER, Wilson RK, Fulton R, Kucaba
TA, Wagner-McPherson C, Barbazuk WB, Gregory SG, Humphray
SJ, French L, Evans RS, Bethel G, Whittaker A, Holden JL,
McCann OT, Dunham A, Soderlund C, Scott CE, Bentley DR, Schuler
G, Chen HC, Jang W, Green ED, Idol JR, Maduro VV, Montgomery
KT, Lee E, Miller A, Emerling S, Kucherlapati, Gibbs R, Scherer
S, Gorrell JH, Sodergren E, Clerc-Blankenburg K, Tabor P,
Naylor S, Garcia D, de Jong PJ, Catanese JJ, Nowak N, Osoegawa
K, Qin S, Rowen L, Madan A, Dors M, Hood L, Trask B, Friedman
C, Massa H, Cheung VG, Kirsch IR, Reid T, Yonescu R, Weissenbach
J, Bruls T, Heilig R, Branscomb E, Olsen A, Doggett N, Cheng
JF, Hawkins T, Myers RM, Shang J, Ramirez L, Schmutz J, Velasquez
O, Dixon K, Stone NE, Cox DR, Haussler D, Kent WJ, Furey T,
Rogic S, Kennedy S, Jones S, Rosenthal A, Wen G, Schilhabel
M, Gloeckner G, Nyakatura G, Siebert R, Schlegelberger B,
Korenberg J, Chen XN, Fujiyama A, Hattori M, Toyoda A, Yada
T, Park HS, Sakaki Y, Shimizu N, Asakawa S, Kawasaki K, Sasaki
T, Shintani A, Shimizu A, Shibuya K, Kudoh J, Minoshima S,
Ramser J, Seranski P, Hoff C, Poustka A, Reinhardt R, Lehrach
H. A physical map of the human genome. The International Human
Genome Mapping Consortium. Nature. 2001 Feb 15;409(6822):934-41.
Ross M.T., LaBrie, S., Mc Pherson, J. & Stanton, V.P.
In Current Protocols in Human Genetics (eds Dracopoli,m N.
C. et al.) 5.6.1-5.6.5 (Wiley, New York, 1999)
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