Southern blot reverse transfer protocol
(for +charged Nylon membranes)
DNA digestion
10 to 15ug of DNA
2-3 uL High activity Enzyme
4 uL 10X Enzyme Buffer
0.4 uL 100X BSA
1.6 uL 100 mM Spermidine
X uL Water (DNase free)
40 uL Total
Run gel overnight at 20-25 volts
**Make sure you run a NEB 1 kb ladder with your samples. You can detect the
ladder following Southern hyb.
1) After running gel, stain in 10 ug/ml EtBr in ddH2O for 30min.
2) De-stain gel in fresh ddH2O for 30min.
3) Photograph with fluorescent ruler
4) Place gel in container and add 0.25M HCL (10.8ml HCL, ddH2O to
500ml). Shake for 20 min.
5) Quickly rinse gel 3x with dH2O
6) Wash gel in 0.4N NaOH (16g NaOH, ddH2O to 1L) and shake in
500mls for 20min.
Assemble southern blot as follows:
3 sheets
1 sheet
5 sheets
20 sheets
0.4N NaOH
Notes:
1) Place stack tray on gel caster to make sure it’s level!!!
2) Lay gel well side up.
3) Pre-wet membrane in transfer buffer for 5 min. Don't let it dry out.
4) Place parafilm around the gel and between the upper and lower 3mm
papers to prevent wicking through the paper. You want it to wick through
the gel.
5) Pre-wet the surface of the agarose gel before placing pre-soaked 3mm
paper on top.
6) 3mm Whatman paper pre-soaked in transfer buffer should be saturated by
rolling a pasteir pipet over paper immersed in buffer.
7) Perform the same trick to remove bubbles between the gel and the
membrane.
8) Cover the apparatus with Saran wrap to prevent evaporation.
7) Once assembled, place 150-200mls of transfer buffer into reservoirs.
8) Transfer O/N.
9) Neutralize the membrane in 0.2M Tris-HCL pH 7.5; 2xSSC (100mls 1M
Tris-HCL pH 7.5; 50mls 20x SSC) twice for 10min each.
10) Place membrane between two sheets of 3mm paper.
11) Bake at 80C for 1 hr.
12) Leave membrane on bench to dry.
13) Store at RT in plastic bag or hybridization tubes.
Probe labelling and nucleotide removal:
Materials:
Invitrogen
Random primers DNA labeling system
18187-013
GE sciences
Illustra ProbeQuant G-50 Micro Columns
28-9034-08
[α-32P]dCTP, 3000 Ci/mmol, 10 μCi/μl
EasyTides
Method:
1. Denature 25 ng of DNA (optionally along with 1ul of 1pg/ul DNA ladder)
dissolved in 5-20 μl of distilled water in a microcentrifuge tube by heating for 5
min in a boiling water bath, then immediately cool on ice.
2. Perform the following additions on ice:
2 μl dATP solution 2 μl dGTP solution 2 μl dTTP solution
15 μl Random Primers Buffer Mixture
5 μl (approximately 50 μCi) [α-32P]dCTP, 3000 Ci/mmol, 10 μCi/μl
Distilled water to a total volume of 49 μl Mix briefly.
3. Add 1 μl Klenow Fragment, mix gently but thoroughly, and centrifuge briefly.
4. Incubate at 25°C for 1 h.
5. Add 5 μl Stop Buffer.
6. purify labeled probe using G50 micro column
7. Remove 30ul of labeled probe (save remainder in freezer).
8. Boil probe and salmon sperm dna for 5 minutes in dry heat block
9. put on ice for 5 minutes
10. Add probe and 200ul ssDNA together, then add to the bottom of the hyb
bottle (to pre-hyb solution), cap bottle, swirl to mix.
11. Hyb overnight
Hybridization:
Materials:
20xSSC
20% SDS (w/v)
Rapid-hyb buffer (Amersham)
OR
Church Hyb buffer:
NaPO4, pH 7.2 0.5 M (from 1 M stock)
SDS 7%
EDTA 1 mM
BSA 1%
dH20
Wash 1 (RT):
2xSSC, 50mls 20X SSC
0.1%SDS, 2.5mls 20% SDS
to 500mls with milliQ H2O
Wash 2 (65C):
2xSSC, 50mls 20X SSC
0.1%SDS, 2.5mls 20% SDS
to 500mls with milliQ H2O
Wash 3 (65C)
0.1XSSC, 5mls 20x SSC
0.1%SDS, 5mls 20% SDS
to 1L with milliQ H2O
Hyb:
- Pre heat hyb buffer plus in a 65C water bath.
- boil 200ul of salmon sperm dna for 5 mins, cool on ice, add to hyb buffer
1) Prewet the membrane for 5 mins in 1 x SSC, 0.1% SDS
2) Place membrane in hyb buffer and pre-hyb at 65C for 2 hrs to overnight.
3) Add labeled probe to pre-heated hyb buffer either new buffer or add to
bottom of hyb tube
4) Mix by inversion
5) Hybridize overnight at 65 C.
6) After the hybridization, empty hyb buffer in radioactive waste.
7) Add ~25 mL wash 1.
8) Wash at RT for 10 min with agitation.
9) Remove wash buffer #1 and replace with wash buffer #2.
10) Place tray @65C and incubate for 15 min.
11) Remove wash buffer #2 and replace with wash buffer #3.
12) Incubate @65C for 20 min.
13) Remove wash buffer #3 and replace with fresh wash buffer #3
14) Incubate @65C for 20 min.
15) Wrap membrane with Saran wrap.
16) Autoradiograph