Holmes, Dawn 1-6
Southern Blot
Reagents:
1. Gel soaking solution (0.25M HCl)
a. Work in fume hood
b. Add acid to water
c. Measure out 490ml Milli-Q water
d. Add 10ml concentrated HCl
2. Transfer solution (1.0M NaCl, 1.6M NaOH)
a. Measure out 800ml Milli-Q water
b. 58.44g NaCl
c. 16g NaOH
d. Adjust volume to 1L.
3. 0.5M NaH2PO4, (monobasic) pH7.2
a. Measure out 400ml Milli-Q water
b. Warm water
c. Add 69g NaH2PO4
d. Cool to room temperature
e. Adjust pH to 7.2 with NaOH
f. Adjust volume to 1L.
4. Hybridization solution (0.5M NaH2PO4, 1mM EDTA, 7% SDS)
a. Measure out 200ml Milli-Q water
b. Warm water
c. Add 34.5g of NaH2PO4
d. Cool to room temperature
e. pH to 7.2 with NaOH
f. Add 1ml of 0.5M EDTA
g. Add 35g of SDS.
h. Adjust volume to 500ml.
5. Wash I Buffer (40mM NaH2PO4, EDTA, 5% SDS)
a. Measure out 800ml Milli-Q water
b. Add 80ml of 0.5M NaH2PO4
c. Adjust pH to 7.2
d. Add 2ml of 0.5M EDTA
e. Add 50g of SDS.
f. Adjust volume to 1L.
6. Wash II Buffer (40mM NaH2PO4, 1mM EDTA, 1% SDS)
a. Measure out 800ml Milli-Q water
b. Add 80ml of 0.5M NaH2PO4
c. Adjust pH to 7.2
d. Add 2ml of 0.5M EDTA
e. Add 10g of SDS.
f. Adjust volume to 1L.
7. TE + 20mM NaCl
8. Sephadex G-25 (stored in the fridge in hot lab) for purifying probes
a. Swell sehadex in excess TE+ 20mM NaCl.
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b. Heat at 65oC with occasional stirring for several hours to degas.
c. Store at 4oC with 0.2% azide.
Running Gels for Southern Blots:
1. Load approximately 2.5 μg of genomic DNA per lane.
2. Load less that 100 ng of positive control DNA onto the gel
3. It is a good idea to load the positive on one side of the molecular weigh standards, and
the samples on the other or to skip a lane between the positive and the samples.
4. If you have the space load a visible amount of positive in one lane to be cut off and small
amount in the next lane for the blot.
a. This can be used to measure your positions.
5. Run the gel slowly, or you will not get good resolution.
6. About 6 hr at ~50 V.
7. Run the dye to about three cm from the bottom of the blot.
a. You will not loose small products.
b. If you are worried, check the position of the markers.
Transferring DNA into Zeta Probe Membrane
1. Stain gel
2. Photograph markers with ruler to that you can determine their position on your blot later.
3. If necessary cut gel to appropriate size.
4. Cut a piece of zeta probe so that it is the same size as the gel.
a. Be careful to wear gloves at all times and use clean forceps.
5. Cut off top right hand corner of membrane
a. You will later put cut corner over top right hand corner of gel
6. Mark where wells will be with ball point pen.
7. Soak zeta probe in clean dH2O until ready to assemble blot.
8. Soak gel 15 minutes in 0.25 M HCl.
9. Rinse gel rapidly 3 times in dH2O.
10. During 15 minute incubation prepare for transfer.
a. See pictures:
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Probe Preparation:
1. We use the NEB blot kit
2. Digest vector to excise insert to be used as probe, or amplify relevant PCR product.
3. Gel purify the insert or PCR product.
4. Determine the concentration of your insert in solution by running a gel.
a. Load various concentrations of insert and molecular mass markers
b. Determine a rough concentration of the insert by comparing the brightness of the
bands.
c. Too little or too much insert will greatly decrease the efficiency of incorporation
of 32P into the probe.
5. In a screw cap eppendorf tube mix:
a. 25ng of probe
Holmes, Dawn 4-6
b. Enough dH2O to bring to a volume of 32 µl
6. Boil for 5 minutes
7. DO NOT SPIN
8. Transfer tube immediately to ice.
9. Chill for 5 minutes
10. While tube is on ice add:
a. 5μl 10x Labelling buffer
b. 2μl of dGTP
c. 2μl of dATP
d. 2μl of dTTP
e. 5μl α32P dCTP
f. Sometimes we use α32P dATP and substitute 2μl dCTP for dATP.
11. Mix with pipet-man.
12. Spin briefly.
13. Mix again.
14. Add 1 μl if Klenow
15. Incubate at 37˚C for 1 hour.
16. Add 5 μl of 0.2M EDTA to stop reaction
17. Place on ice
Cleaning Probe on Spin Column:
1. Separates unincorporated nucleotides from the probe.
2. Unincorporated nucleotides will remain trapped in the resin and the probe will elute.
3. Add 50 μl of TE+NaCl to probe
4. Stuff a little bit of glass wool into a 1 ml syringe
5. Fill syringe with a 50:50 slurry of G-25 and TE + NaCl.
6. Place syringe in 15 ml centrifuge tube. (see picture)
7. Spin in clinical centrifuge at approximately 1,000-2,000 xg for 2 minutes.
8. Discard liquid in bottom of 15 ml tube.
9. If the bottom of the syringe was sitting in liquid after the spin, spin again.
10. After spinning, the matrix should reach between the 0.5ml and 0.7 ml mark on the
syringe.
11. Place new screw cap eppendorf at bottom of 15 ml tube.
12. Place syringe column back into tube.
13. See picture.
14. Load probe onto the syringe column.
15. Spin in clinical centrifuge at approx. 1,000-2,000 x g for 2 minutes.
16. Probe will be in liquid in eppendorf.
17. Unincorporated nucleotides will remain in the spin column which is screaming hot.
18. Discard spin column,
19. If 32P dCTP incorporation is good the probe will also be screaming hot.
20. If probe is used the same day.
21. Store on ice.
22. Probe can be stored in freezer for 2 to 3 weeks.
Holmes, Dawn 5-6
Probe Denaturation (for Northerns):
1. Want to use about 1/3 of probe (~30 μl) per blot.
2. Thaw probe and aliquot appropriate amount in new screw cap tube.
3. Boil 5 min
4. Put on ice for 5 min
Probing Southern Blot:
1. Prehybridization:
a. Prewarm Hybridization Solution to 65˚C.
i. Make sure all the SDS goes into solution.
b. Prewarm hybridization oven to 65˚C.
c. Put zeta-probe into hybridization tube
d. Add 10-15 ml of warm hybridization solution.
e. Prehybridize at 65˚C for at least one hour.
2. Probe Denaturation:
a. To 100 μl of probe add
i. 5 μl of 4N NaOH
ii. 10ml of 10% SDS
b. Mix
c. Boil 5 minute
d. Transfer immediately to ice and chill for 5 minute.
e. Spin briefly to get liquid off sides of tube.
f. Immediately place back on ice.
3. Hybridization:
a. Add 25-50 μl of probe to hybridization tube containing the blot.
i. Depending upon how hot and how new it is.
ii. The remaining probe can be stored at -20˚C for future use.
b. Make sure gasket on caps for hybridization oven are dry before sealing tube.
i. Otherwise probe may leak out.
c. Incubate overnight at 65˚C in hybridization oven.
4. Washing:
a. Prewarm Wash I buffer and Wash II buffer to 65˚C.
b. Pour off hybridization solution into liquid 32 P waste bottle.
c. Give the blot a quick rinse with Wash I buffer.
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d.
e.
f.
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l.
Pour the liquid into 32P waste bottle.
Fill tube ~1/3 full with Wash I buffer.
Incubate for 30 minutes at 65˚C in hybridization oven.
Pour liquid into 32P waste bottle.
Wash once more for 30 minutes at 65˚C with Wash I buffer.
Wash twice for 30 minutes at 65˚C with Wash II buffer.
After last wash blot to dry briefly on Whatman paper.
Wrap in Saran wrap
Expose to film or phosphoimager plate.