Preparing Y-Chromosome Probe

If you are starting with an aliquot of 2-4 µl template DNA, amplify the DNA several (2-3) times by DOP-PCR to generate Y chromosome template. This will be your Y chromosome template stock. Then each time you make probe use DNA from this stock for another batch (8 reaction) DOP-PCR and subsequent labeling. Do not repeatedly amplify DOP-PCR as this can bias the oligos generated by selecting for a subpopulation of oligos that amplify particularly well.

We usually generate Y probe in bulk by making a master mix for 10 PCR reactions. We then make 8 DOP-PCR reactions, have one no DNA control and 1 excess reaction for pipetting error. This bulk reaction gives us 1.6 mls of Y probe, roughly enough to stain 160 slides.

(degenerate oligonucleo primed-polymerase chain reaction)

PCR reaction, 50 µl total volume

PCR Reaction Reagents

Reagent	1 Reaction	10 Reactions
Water	40.3 µL	403 µL
10x PCR buffer	5 µL	50 µL
10 mM dNTP	1.2 µL	12 µL
100 µM 6AI primer	1 µL	10 µL
Taq stock 5U/µl (Roche cat# 1435094)	0.5 µL	5 µL

(Roche cat# 1435094)

Take out 48 µl for no DNA control, place in a labeled 0.2ml tube.

Add DNA

Reaction Reagents

Reagent	1 Reaction	9 Reactions
Mouse Y chromosome DNA	2 µL	18 µL

Pipette 50 µl into 8 tubes

6AI Primer = CCG ACT CGA GNN NNN NTA CAC C where N is an equal concentration of all nucleotides

PCR Cycling Conditions

PCR Cycle	Denaturing	Annealing	Extension
1-2 cycles	45 sec / 94°C	45 sec / 15°C	12 min / 37°C
5 cycles	40 sec / 94°C	45 sec / 37°C	4 min / 66°C
24 cycles	40 sec / 94°C	45 sec / 54°C	4 min / 66°C

For bulk DOP-PCR, pipet all the reactions together after cycling. Then run 5 µl pooled DOP-PCR reaction and 5 µl no-DNA control on a 2% agarose gel alongside a 100 bp marker. The DOP-PCR reaction produces a smear that runs from approximately 300 to 2.5 kb. Next ethanol precipitate the pooled DOP-PCR DNA overnight by adding ¹⁄₁₀ the volume of 3M NaOAc, vortexing and then adding 2.5x the volume 100% ethanol and leaving at -20°C ON. The next day centrifuge the solution at 14,000 rpm for 30 min to pellet the DNA. Next wash the pellet in 70% ethanol and air-dry. Resuspend the pellet in the starting volume of distilled water (50 µl per PCR reaction, 400 µl for 8 reactions pooled).

DIG-Nick Translation

The next step involves digesting the Y chromosome template DNA (DOP-PCR reaction) into smaller pieces and labeling these pieces with digoxigenin. This is done using the DIG-Nick translation kit (ROCHE cat# 1745876). Take out 5-20 µl of newly resuspended DOP-PCR and set aside.

Reaction Reagents

Reagent	1 Reaction	8 Reactions
Y Chromosome Template (DOP-PCR)	approx. 50 µl	approx. 400 µl
DIG-Nick Kit mix	12.5 µl	100 µl

This is now your DIG-Nick reaction solution. Incubate at 15 °C for 90 min to 2 hrs. Put the reaction on ice and run the reaction on a 2% agarose gel alongside the 5 µl of DOP-PCR you set aside earlier and some 100bp marker. You expect to see smear of fragments ranging from 150-500 bp. If this is the case, stop the reaction. If the smear still contains larger sized fragments, return the reaction to 15°C and check again on a 2% agarose gel every 10-20 min until the reaction contains the appropriate fragment sizes.

To stop the reaction:

Reaction Reagents

Reagent	1 Reaction	8 Reactions
DIG-Nick reaction	62.5 µl	500 µl
0.5M EDTA	4 µl	32 µl

and incubate at 65°C for 10 min.

Ethanol precipitation with carrier and blocker DNA

Aliquot the DIG-Nick reaction by placing 100 µl of it into several 1.5 ml eppendorfs. Scale up or down as necessary.

Reaction Reagents

Reagent	1 Reaction
DIG-Nick reaction	100 µl
Sonicated Salmon sperm 10 mg/ml (Stratagene cat# 21190)	4 µl
Cot-1 DNA 1 mg/ml (Invitrogen cat# 18440-016)	100 µl
3 M NaOAC (then vortex)	26 µl
100% ethanol	800 µl

Vortex again and leave ON at –20°C. Centrifuge at 14,000 rpm for 30 min to pellet the DNA. Next wash the pellet in 70% ethanol and air-dry. Resuspend the pellet in 100 µl formamide DI (deionized). Make sure the pellet is completely resuspended by heating the solution to 42°C for 10 min or higher if necessary. Then add an equal volume of hybridization buffer and store the probe at -20°C.

Hybridization Buffer

For 20 mls:
8 mls 50% Dextran Sulfate - end conc. 20% Dextran Sulfate
4 mls 20x SSC - end conc. 4x SSC
8 mls H2O