TITLE: Southern blot
AUTHORS: Oriol Busquets, Frank Soldner

[DESCRIPTION]
This protocol describes a southern blot analysis adapted from standard protocols (https://cshprotocols.cshlp.org/content/2021/7/pdb.top100396#cited-by) to validate the structural integrity and exclude the loss of heterozygosity (LOH) at a genomic locus of interest (PRKN, DJ1, FBXO7 and SYNJ1) following CRISPR/Cas9 or prime editing (PE) based genome editing experiments in human pluripotent stem cells. 

Protocol Overview
A. Buffers
B. Generation of Southern blot probes
C. Cell lysis, gDNA extraction and purification
D. gDNA digestion
E. Agarose gel electrophoresis and transfer
F. Membrane blocking and probing
G. Wash and exposure
H. Exposition

Initial Comments

This protocol requires the use of radiolabeled nucleotides and consequently, requires the handling of radioactive materials. Follow your institution's training, regulations and procedures to ensure proper handling of these hazardous materials and ensure researcher’s safety.

A list of reagents and relevant vendor information can be found in the table listed under the materials tab.

Oligonucleotide sequence table

 


[STEPS]
SECTION: Buffers
1. Prepare the following buffers:
SECTION: Generation of Southern blot probes
2. 
 

 

 

To generate southern blot probes use AccuPrime‱ Taq DNA Polymerase, high fidelity; ThermoFisher.12346086 on WIBR3 (parental; RRID:CVCL_9767) gDNA using primers described above.
SECTION: Buffers
1.2. 20X SSC buffer: 3M NaCl, 0.3M sodium citrate.
SECTION: Buffers
1.3. Depurination buffer: 0.25M HCl.
SECTION: Buffers
1.1. Lysis buffer: Tris-Cl pH 8.5 (100mM), EDTA (5mM), NaCl (200 mM), SDS (0.2%) supplemented  with proteinase K (100 µg/ml) freshly thawed from frozen stocks. 
SECTION: Buffers
1.4. Denaturation buffer: 0.5M NaOH, 1.5M NaCl.
SECTION: Buffers
1.5. Church buffer: 1 mM EDTA, 0.5M NaPO4 (pH7.2; critical to check pH), 7% SDS, 1% (w/v) bovine serum albumin (add BSA fresh before usage).
SECTION: Generation of Southern blot probes
2.1. Reaction setting: gDNA template (100-200 ng) + 1 µl sense primer (10 µM) + 1 µl anti-sense primer (10 µM) + 5 µl AccuPrime Buffer II + 0.2 µl AccuPrime‱ Taq DNA Polymerase, High Fidelity  (1U) + molecular grade water (to 50 µl).
SECTION: Generation of Southern blot probes
2.2. Thermocycler settings: (94 C - 2min) + 30 cycles (94 C - 30 sec + 58 C - 30 sec + 68 C - 1 min/kb) + (68 C - 10 min) + (16 C - ∞).
SECTION: Generation of Southern blot probes
3. After the reaction is complete, confirm the purity of the probe (PCR product) by running either a small sample or the entirety of it on an agarose gel (0.8%). Purify the gel band or remaining PCR product by using E.Z.N.A.‱ Gel Extraction Kit by Omega Bio-Tek; Omega Bio-Tek; D5000 (follow manufacturer’s instructor on how to prepare each of the necessary reagents).
SECTION: Cell lysis, gDNA extraction and purification
4. 
 

Remove media from hESC cultures and add Lysis buffer (1 ml/well in 6 well plate and 0.5 ml/well in 12 well plate) and incubate for several hours to   at 37 °C. 
SECTION: Cell lysis, gDNA extraction and purification
5. Transfer up to 750 µL of lysate into a 1.5ml Eppendorf tube and add an equal volume of isopropanol. Vigorously shake the tube (no vortexing to avoid shearing of DNA) until the sample has mixed properly and a white filamentous pellet can be observed inside.
SECTION: Cell lysis, gDNA extraction and purification
6. Centrifuge your sample on a countertop microcentrifuge (Max speed , ⩾13000 x g - 5 min - Room temperature).

SECTION: Cell lysis, gDNA extraction and purification
7. Decant/aspirate the supernatant and add 1 mL of 70% ETOH.
SECTION: Cell lysis, gDNA extraction and purification
8. Briefly shake the tube (no vortexing to avoid shearing of DNA) to wash the pellet in 70% ETOH.
SECTION: Cell lysis, gDNA extraction and purification
9. Centrifuge your sample on a countertop microcentrifuge (Max speed, ⩾13000 x g - 5 min - Room temperature).

SECTION: Cell lysis, gDNA extraction and purification
10. Decant supernatant, aspirate any residual liquid from the tube and  leave the cap open to air dry for 10 min-20 min until pellet starts to become translucent (depending on pellet size time and color may vary). Do not overdry the sample as this could prevent complete resuspension of gDNA. 
SECTION: Cell lysis, gDNA extraction and purification
11. Resuspend the pellet in TE buffer and allow it to slowly solubilize for at least 720 min-24 hours at 56 °C before use.
SECTION: gDNA digestion
12. Restriction digestion of genomic DNA should be optimized for the enzyme being used.
SECTION: gDNA digestion
12.1. Reaction setting: gDNA (10-15 µg) + Restriction  Enzyme (4 U per 1 µg DNA, not more than 5 µl enzyme/reaction - check table above to see gene/probe/enzyme combinations used) + 5 ul Buffer (rCutSmart - buffer may vary depending on enzyme being used) + molecular grade water (to 50 µl).
SECTION: gDNA digestion
12.2. Incubate samples at 37 °C for 360 min to 360 minincubation (some enzymes may require alternative temperature settings for their efficient performance).
SECTION: Agarose gel electrophoresis and transfer
13. Digested gDNA should be run on a 0.8% agarose gel with ethidium bromide to separate the sample. A 1 kB ladder should be added next to the samples to be able to discern the molecular weight of bands detected (1/5-1/10 dilution of the marker may be necessary to prevent its signal from overwhelming the blot). Gel size, separation length and electrophoresis conditions should be optimized for each digest. Electrophoresis voltage should be kept rather low to ensure proper migration and avoid overheating of gel.
SECTION: Agarose gel electrophoresis and transfer
14. Once the electrophoresis is complete, carry your gel to a UV reader and collect an image that can be used as reference to locate sample tracks and marker distribution. Trim the areas of the gel above the wells with a razor blade. Make sure to keep track of the wells location. Marks can be made on the edges to be able to confirm the proper orientation.
SECTION: Agarose gel electrophoresis and transfer
15. Depurination: in order to reduce DNA fragment size and increase transfer efficiency, gels can be depurinated to create nicks in the strands in the gel. For depurination, incubate gel for 15 min in depurination buffer (0.25M HCl) with gentle shaking - excess shaking may cause the gel to break.
SECTION: Agarose gel electrophoresis and transfer
16. Denaturation: incubate the gel in a denaturation buffer for 45 min to 1 hour (0.5M NaOH, 1.5M NaCl). This same solution is used for the transfer. Usual amounts used are ~2L/blot for both denaturation and transfer steps. Prepare more or less as required.
SECTION: Agarose gel electrophoresis and transfer
17. DNA transfer to membrane: The DNA transfer is accomplished by the creation of an osmotic potential from one end to the other of the transfer structure. To set up the transfer assembly (see Figure 1 in Green and Sambrook; https://cshprotocols.cshlp.org/content/2021/7/pdb.top100396#cited-by) first prepare a tray (buffer reservoir) and place a glass plate on top in a sideways position. To assemble the transfer assembly, stack the following components from bottom to top on the glass plate: 
SECTION: Agarose gel electrophoresis and transfer
17.1. Whatman paper sheets are placed on top of the glass so that there is an overhang going into the buffer reservoir tray underneath. Soak the Whatman paper sheets with denaturation buffer. 
SECTION: Agarose gel electrophoresis and transfer
17.2. Place the gel on top of the Whatman paper in an inverted position so that the upper side where the wells were generated on the agarose gel are on its underside against the paper. This will ensure a flat surface for the DNA transfer increasing its efficiency.
SECTION: Agarose gel electrophoresis and transfer
17.3. Place a pre-wetted (denaturation buffer) nylon membrane (Amersham Hybond XL) of similar size to the gel on top of the gel. To prevent bubbles between the gel and the membrane use a serological pipette as a roller to remove any bubbles.
SECTION: Agarose gel electrophoresis and transfer
17.4. Place several pre-wetted (denaturation buffer) Whatman paper of similar size to the gel on top of the nylon membrane. Use a serological pipette as a roller to remove any bubbles.
SECTION: Agarose gel electrophoresis and transfer
17.5. Place parafilm strips on the edges around the gel on all sides to ensure that no buffer can pass through the gel before reaching the nylon membrane (to prevent buffer shortcuts and increase osmotic potential).
SECTION: Agarose gel electrophoresis and transfer
17.6. Place sufficient stack of dry paper towels on top of the transfer assembly and weighted down with a tray and mild pressure (not too much as to damage the gel) to ensure equal transfer through the entire transfer assembly.
SECTION: Agarose gel electrophoresis and transfer
17.7. Fill the buffer reservoir tray at the bottom with sufficient denaturation buffer to allow a DNA transfer for 360 min to 360 min. 
SECTION: Membrane blocking and probing
18. Take apart the transfer assembly. Membranes have directionality and it can only be probed from one side, mark the membrane with pencil to keep track of its orientation and wash the membranes with 2xSSC buffer (0.3 NaCl, 0.03M sodium citrate; 3 times 10 min).
SECTION: Membrane blocking and probing
19. Heat-up the hybridization chamber and cylinders. For each membrane, preheat 2 tubes containing  20 mL of Church buffer (1 mM EDTA, 0.5M NaPO4 (pH7.2; critical to check pH), 7% SDS, 1% (w/v) bovine serum albumin [BSA}, add BSA fresh before usage ) to 62 °C-65 °C.
SECTION: Membrane blocking and probing
20. Put the membrane into the cylinders, ensuring the side with the transferred DNA is facing inwards and add in 20 mL of preheated Church buffer . Incubate inside the hybridization chamber for at least 60 minto block the membrane efficiently (constant rotation; 62 °C-65 °C).
SECTION: Membrane blocking and probing
21. In the meantime, prepare the radiolabeled-probes (50 ng DNA/probe) using the Prime-It II Random Primer Labeling Kit (300385-52; Agilent) and dCTP, [α-32P]-3000Ci/mmol 10mCi/ml EasyTide (BLU513H100UC) following the instructions of the vendor.
SECTION: Membrane blocking and probing
22. Add the radiolabeled-probe into a resin-based column (Ilustra ProbeQuant G-50 Micro Columns) to purify it and add it to the second tube with preheated Church buffer. Remove the blocking solution and add the one containing the radiolabeled probe to the cylinder (60 minincubation, constant rotation; 62 °C-65 °C).
SECTION: Wash and exposure
23. The next steps consist of washes to remove excess  and nonspecific bound radiolabeled probes. Washes are done using increasing stringency SSC buffer solutions in the range of 2X (low stringency) to 0.2X (high stringency) + constant 0.2% SDS in all of them. Recommended time is 5 min-10 min/wash. Use a Geiger counter in between washes to track radioactivity levels. Washes may be done inside the hybridization chamber canister or inside a sealed container under constant rotation/shaking and  62 °C-65 °Cconditions.
SECTION: Wash and exposure
24. Repeat these steps and increase stringency as necessary until the radioactive signal becomes modest. Bands may be detected by the appearance of sudden spikes.
SECTION: Wash and exposure
25. Put the membranes into plastic foil and seal the edges to ensure the membranes don’t dry.
SECTION: Wash and exposure
26. Put it inside a cassette and expose it to a film and leave for 1440 min to 72 hours at -80 °C(Exposition time may vary and lengthen), when exposed and seeing the signal adjust time as necessary. The blot will be good to be used for about 2 months, but signal will be lost over time.
If too much background signal is observed, blots can be washed again as previously stipulated and re-exposed to films until the desirable signal is obtained.

 

 



SECTION: Exposition
27. A film developing system was used to obtain the signal in the film.