Southern blot test for the Fragile X syndrome
Digestion of genomic DNA
1.
Add 7 µg genomic DNA (35 µl of 200 ng/µl) to a labeled 1.5 ml microcentrifuge
tube. Bring total volume to 35 µl with sterile distilled H2O.
Use as control samples a female sample with a normal and a full mutation allele
and a female sample with a normal and premutation allele.
2.
Prepare reaction mix in a 1.5 ml microcentrifuge tube by combining the
following:
Hind lll + Eag l: 4.75 µl aqua dest
5 µl 10x SuRE/Cut Buffer H (Roche)
3 µl 50 mM spermidine
1.25 µl Hind lll (Roche, 40 U/µl)
1µl Eag l (Biolabs, 50 U/µl)
Mix well
Prepare enough mix for n + 1 samples
3.
Transfer 15 µl reaction mix to each DNA sample and mix well.
4.
Incubate digestions o/n at 37 °C.
Testing of digestions on test gel (Optional)
5.
Prepare a 0.7% agarose Boehringer MP testgel.
6.
Combine 5 µl of each digestion product with 5 µl 2x Ficoll loading buffer.
7.
Load samples on 0.7% testgel in 1x TBE buffer with Etbr and run for 2-3 hours
at 80 V.
8.
Make a picture under UV light and look if digestions were complete.
Separate digestion products on 0.7% blotgel
9.
Prepare a 0.7 % Boehringer MP agarose blotgel.
10. Add 5 µl 10x Ficoll loading buffer to 45 µl of each digestion product
11. Prepare a marker by combining the following:
0.5 µl 1 kb ladder (InVitroGen)
5 µl DNA molecular weight marker II, DIG labeled (Roche)
30.5 µl 10 mM Tris/1 mM EDTA pH 7.5
4 µl 10x Ficoll loading buffer
12. Load samples on 0.7% agarose blotgel in 1x TBE buffer.
13. Electroforese o/n at 45 V untill the OrangG front has run about 20 cm from the
slots.
Photography of the blot gel (Optional)
14. Stain the gel in 1x TBE buffer with ethidiumbromide and make a picture of it.
Southern Blotting
15. Place gel 15 minutes in 0.25 M HCl with gentle shaking.
16. Place gel 30 minutes in denaturation buffer with gentle shaking.
17. Place gel 2 x 15 minutes in neutralization buffer with gentle shaking.
18. Fill a tray with 1 liter 20x SSC. Make a platform using a glassplate and cover it
with a 20 x 40 cm S&S GB004 gel blotting paper, saturated with 20x SSC.
Place parafilm on the sides of the blotting paper to prevent the blotting buffer
being absorbed directly into the paper towels above.
19. Place the gel on the wick and avoid trapping air bubbles beneath it.
20. Cut a sheet of Boehringer Nylon membrane to the exact size of the gel, mark
with a pencil and place on top of the gel. Avoid trapping air bubbles beneath the
membrane. Use a pipette and roll over the membrane to make assure good
contact between gel and membrane.
21. Place three sheets of GB002 paper, wetted with 20x SSC on top of the
Boehringer Nylon membrane.
22. Place a stack of grey paper towels on top of the S&S GB002 paper.
23. Place a glass plate on top of the paper towels and put a 0.75-1 kg weight on
top. Allow transfer to proceed o/n.
24. Dismantle the blot apparatus after the DNA transfer and rinse the membrane for
a few seconds in 2x SSC. Let the membrane dry on filter paper.
25. Fix the DNA on the membrane by baking the membrane 15-30 minutes at 120°
C or 2 hours at 80 °C.
(Pre)hybridization of membranes
(Pre)hybridization of membranes can be carried out in hybridization bags or even
better a hybridization oven.
26. Put 20 ml of DIG Easy Hyb solution in a roller bottle and put the bottle in a
hybridization oven at 42 °C and 10 rpm (rotations per minute).
27. Pre-wet a piece of mesh in 2x SSC, put it on a piece of Saran Wrap and place
the Boehringer Nylon membrane with the DNA side up on the mesh. Roll mesh
and membrane up and put it in the hybridization bottle with the pre-heated DIG
Easy Hyb hybridization solution. Let it hybridize for at least 1 hour at 42 °C and
10 rpm.
28. Denature 400 ng digoxigenine labeled probe for 5 minutes at 95°C. Quick chill
denatured probe on icewater.
29. Add the denatured probe to 20 ml fresh at 42 °C pre-heated DIG Easy Hyb
hybridization solution.
30. Replace the prehybridization solution in the roller bottle with the hybridization
solution. Hybridize o/n at 42 °C.
31. Throw away the hybridization solution and continue with the washing procedure.
Washing of the membranes
The washings of the membrane can also be done in the roller bottle in the
hybridization oven. If you just start with the DIG method, it is better to do the washes
in trays, because in bottles there’s a greater risk of background.
32. Wash the membrane 2 x 5 minutes with 50 ml 2xSSC/0.1%SDS at room
temperature.
If the washing is carried out in the hybridization oven, we put the RT 2x SSC/0.1%
SDS solution in the bottles and during these washing steps we raise the temperature
of the oven to 65°C. As the 65°C washes are starting, then the oven is already at the
correct temperature.
33. Wash the membrane 2 x 15 minutes in 1x SSC/0.1%SDS at 65 °C.
Detection of the digoxigenine labeled probe
The detection procedure is done in a tray. Be sure to use clean trays, after cleaning
also rinse with aqua dest. We use Nalgene Nunc dishes (245x245x25 mm) These
can easily contain a blot of 20x20 cm.You can re-use them.
Steps 34 till 45 are done under gentle agitation and at room temperature..
34. Equilibrate membrane 1-5 minutes in 100 ml 1x Washing buffer.
35. Incubate membrane 60 minutes in 200 ml 1x Blocking solution.
36. Take the membrane out of the 1x Blocking solution and put it on a piece of
SaranWrap. Add 10 µl Anti-digoxigenine-AP-conjugate to the 1x Blocking
solution, shake well and put the membrane back in this solution for 30 minutes.
Before pipetting the antibody solution you should centrifuge this solution for 5
minutes at 12000 rpm to get rid of a possible precipitate.
37. Discard the antibody solution and wash the membrane in a clean tray filled with
1x Washing buffer for 15 minutes.
38. Wash the membrane again for 15 minutes in 1x Washing buffer.
39. Equilibrate the membrane 1-5 minutes in 1x detection buffer.
40. Dilute 15 µl CDP star solution with 1500 µl 1x detection buffer.
41.
Place the membrane between two sheets of a cutted plastic bag. Lift the top
sheet and pipette the diluted CDP star solution on top of the membrane. Lower
the top sheet of plastic. Remove any bubbles present under the sheet. Incubate
the membrane for 5 minutes.
Adding the substrate solution and spread it out evenly over the membrane must be
done quickly to prevent background.
42. Remove excess liquid from under the plastic by wiping with tissue over the
membrane to squeeze out the excess liquid. Close the plastic bag by sealing.
43. Make exposures on X-ray film from 10 and 30 minutes. In most cases these
exposure times will be OK.
10x FLM (Ficoll loading mix)
25% Ficoll
0.1 M Tris pH 7.5
10 mM EDTA
0.25 % Oranje G
10x TBE
900 mM Tris
900 mM Boric acid
25 mM EDTA
1x TBE with Etbr (1x gelbuffer with Etbr)
90 mM Tris
90 mM Boric acid
0.25 mM EDTA
0.1 µg/ml Etbr
25 g Ficoll
10 ml 1 M Tris pH 7.5
2 ml 0.5 M EDTA
250 mg Orange G
To 100 ml in H2O
1090 g Tris
556 g Boric acid
93 g EDTA
To 10 l in H2O
1 l 10x TBE
100 µl 10 mg/ml Etbr
To 10 l in H2O
1x TBE without Etbr (1x gelbuffer without Etbr)
90 mM Tris
1 l 10x TBE
90 mM Boric Acid
9 l H2O
0.25 M HCl
206 ml 37% HCl
To 10 l H2O
Denaturationbuffer
0.5 M NaOH
1.5 M NaCl
Neutralizingbuffer
3.0 M NaCl
0.5 M Tris pH 7.0
200 g NaOH
877 g NaCl
To 10 l in H2O
2630 g NaCl
907.3 g Tris
pH 7.0 with HCl
To 15 l with H2O
Nylon Membranes, positively charged 1 roll (0.3 x 3 m)
Roche Cat. no. 1 417 240
DIG Easy Hyb, 500 ml
Roche Cat. no. 1 603 558
DIG Wash and Block Buffer Set. Contents 2x 500 ml 10x Washing buffer*, 500 ml
10x Maleic acid*, 500 ml 10x Blocking solution and 100 ml 10x Detection buffer*.
Roche Cat. no. 1 585 762. Solutions marked with an * can be made by yourselve.
The blocking solution is also available without buying the complete kit. This can save
money.
Anti-Digoxigenin-AP, Fab fragments
Roche Cat. no. 1 093 274
CDP-Star
Roche Cat. no. 1 685 627
Expand High Fidelity PCR system
Roche Cat. no. 1 732 641
Digoxigenin-11-dUTP, alkali-labile, 1 mM
Roche Cat. no. 1 573 152
DNA Molecular Weight Marker II, DIG labeled
Roche Cat. no. 1 218 590
1 Kb Plus DNA Ladder 250 µg (1µg/µl)
Invitrogen Cat.no. 10787-018
DNA Mass Ladder
InVitroGen Cat. no. 10068-013
Labeling of probe pP2 with Expand HF PCR system (Roche)
If you have probe pP2 = StB12.3 = pfxa7, then you can directly start the PCR
reaction with the DIG-dUTP. If you don’t have this probe you can first start a
normal PCR reaction without DIG-dUTP from genomic DNA with primer 1 and
primer 2. If you have one pure 1 kb band from this reaction, you can use about
0.25 µl of this reactionproduct for the DIG labeling reaction.
Sequence primer 1: CGC CAA GAG GGC TTC AGG TCT CCT
Sequence primer 2: GAG ACT GTT AAG AAC ATA AAC GCG GG
The PCR product is 1000 bp.
Mix together in a PCR reaction vessel:
After pipetting the reagents in a reaction vessel, put the reaction immediately in
the PCR machine.
reagents
H2O
10x Expand buffer
25 mM MgCl2
10 mM dATP
10 mM dCTP
10 mM dGTP
10 mM dTTP
1 mM DIG-11-dUTP
10 µM Primer 1
10 µM Primer 2
template, pP2 insert, 25 pg/µl
Expand HF Enzyme mix (3.5U/µl)
µl
13.65
5
5
1
1
1
0.85
1.5
10
10
0.25
0.75
For best results, always overlay the reaction with minerol oil
Perform the following PCR cycles:
1 cycle:
2 min - 95°C
10 cycles: 30 sec - 95°C
30 sec - 65°C
45 sec - 68°C
20 cycles: 30 sec - 95°C
30 sec - 65°C
45 sec - 68°C +20 sec/cycle
1 cycle: 7 min - 68°C
HOLD - 4°C
Load on 2% agarose gel: 1 µl DIG labeled probe and 2 µl DNA Mass Ladder.
Estimate the concentration of the labeled probe. The labeled probe is stable at 20°C for more than one year. An amount of 50 –100 ng/µl probe can be easily
achieved with this PCR kit.
Roche Molecular Biochemicals do have a DIG Application Manual. This is a
very useful manual with a lot of troubleshooting, tips and tricks, all the used
chemicals, kits, recipes of solutions etc. This manual is also on the web:
http://biochem.roche.com/
In our lab we only use ready to use solutions, because it’s so easy to work with
and because of quality rules. But these are very expensive and it is possible to
make some solutions yourselves. I have marked the Roche solution which you
can easily make with an *. The content of these solutions are in the DIG manual
The Roche company offers DIG Easy Hyb, this is a ready to use hybridization
solution. But they als sell these DIG granules for preparing the hybridization
solution yourselve.
3-3-2003
For more info please contact:
Wout Deelen
[email protected]