Overgo screening of BAC library
(Craig, Pam, Mike; version 1.0; Feb 2004)
Overgos are a pair of oligonucleotides with complementary 3š ends used to make radioactively labeled probes for screening filters by hybridization.
Information on stickleback CHORI filters is available at:
http://bacpac.chori.org/threesp213.htm
Design overgoes using Overgo Maker:
http://www.genome.wustl.edu/tools/?overgo=1
Comments on Overgo design:
We typically isolate stickleback sequence initially by degenerate PCR and RT-PCR using alignments including pufferfish and zebrafish sequences. This gene specific sequence we then input into Overgo Maker. Wešve also successfully isolated BACs by designing Overgos to highly conserved regions without isolating stickleback sequence first.
We recommend BLASTing the suggested Overgo sequences to the pufferfish and zebrafish genome assemblies to assure sequences are not repeat elements (e.g. MER repeats). We also recommend avoiding any homopolymer or repetitive stretches. Sometimes Overgo Maker can recommend an overgo with homopolymer stretches greater than three or containing di- or trinucleotide repeats. In these cases we trim this sequence from the input field and select a different target sequence.
Follow the CHORI protocol for hybridization:
(http://bacpac.chori.org/overgohyb.htm)
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 {-dATP, -dCTP}
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 (Amersham
27-2080-01)
5 ul
100 mM dGTP (Amersham
27-2070-01)
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}. Donšt forget the anchor spots! (see below)
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, (DNA side up) 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 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.
(To
maintain correct orientation and prevent filters from tightly winding during
rotation, roll filters away from you. Hold filters in right hand, place into
bottle in left hand. Place in oven with bottle cap to the left).
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)
Notes/comments on CHORI
protocol:
Recipes for making solutions (these amounts are enough for one BAC screen of 4 bottles, use sterile milliQ water for all):
1 L of 1M sodium phosphate buffer, pH 7.2
mix:
142 g Na2HPO4 (sodium phosphate dibasic)
567 ml H2O
4 ml 85% H3PO4 (phosphoric acid)
bring up to 1 L total volume with H2O
400 ml hybridization solution
800 uL 0.5 M EDTA, (ours is premade and pH 8.0, using this works fine)
200 ml 1 M sodium phosphate buffer, pH 7.2
140 ml 20% SDS
to 400 ml total volume with H2O
500 ml wash buffer B
1 ml 0.5 M EDTA, pH 8.0
20 ml 1 M Na2HPO4, pH 7.2
25 ml 20% SDS
to 500 ml with H20
500 ml wash buffer 2 (make 2 500 ml bottles of this)
37.5 ml 20 X SSC
2.5 ml 20% SDS
to 500 ml with H2O
500 ml wash buffer 3
12.5 ml 20 X SSC
2.5 ml 20% SDS
to 500 ml with H20
-
other notes/comments:
-Donšt forget to add anchor spot overgoes!
details are available at: http://bacpac.chori.org/anchors.htm; anchor spot overgo sequences are:
RPCI941a1-OVF
GTTGCCAAATTCCGAGATCTTGGC
RPCI941a1-OVR
ATCATGTGGCTTCGTCGCCAAGAT
-OLB (overgo labeling buffer) should be less than a year old
- We typically combine 3 pairs of Overgoes along with the anchor spot pair of Overgoes into one 12 inch bottle containing 4 stacked filters (all DNA side up!) separated by nylon filters.
-Dispose of first two washes in hot liquid waste!
-Wrap each filter in saran wrap when washes are done. Carefully roll all bubbles out of wrapped filter with 25 ml pipette. Tape two filters onto used piece of film to support in film exposure cassette. This causes small areas of overlap at one corner of each filter but this is not a problem. Remember to tape fluorescent rulers onto each used piece of film (away from filters) as control signal for autorad. If these are taped on at different angles for each piece of used film, one can uniquely identify each pair of filters after the exposure. Remember to expose these rulers to bright light beforehand so they will fluoresce.
-If youšve combined multiple overgoes, determine which spots correspond to each probe by PCR screening colonies. First determine how many ICE (internet contig explorer) contigs are present in each bottle. Usually many BACs from the filter screen fall into a smaller number of ICE contigs. PCR screening representative BACs from each contig then can greatly facilitate figuring out which spots corrrespond to which probes.
(stickleback ICE info is at: http://cegs.stanford.edu/Physical_map.jsp)