Lab 4: Southern blot and plaque lifts
Notes:
The Southern blot is the most common way to analyse restriction endonuclease-digested DNA to find specific sequences of interest. It uses the fact that denatured nucleic acids will bind to nitrocellulose or nylon membranes. DNA fragments are separated by size on a gel, then transferred to the membrane by capillary transfer or electrophoretic techniques. This immobilizes the DNA fragments, allowing specific sequences to be identified by hybridization with single-stranded probes.
Before capillary transfer, several steps must be taken to prepare the DNA for transfer to the membrane. The gel should first be examined under UV light to confirm DNA is present and a separation of bands has occurred. The gel should not be exposed to UV for too long, as this will damage the DNA.
If the gel contains fragments longer than 1kb, the gel is first soaked in an acid solution, which causes partial depurination. Depurinated sites are cleaved in the alkaline solution used in the next step, which breaks the DNA into smaller fragments. (Fragments greater than 1 kb long are not transferred efficiently by capillary action.)
The gel is then soaked in alkaline solution to denature the DNA, and then neutralized in Tris/NaCl solution. The latter step is optional for nylon blots, which are more resistant to alkaline conditions than nitrocellulose.
The transfer of the DNA to the membrane is done by sandwiching the membrane between the gel and a stack of absorbent paper. The paper absorbs buffer from the gel, pulling DNA fragments along. The DNA binds to the side of the nitrocellulose or nylon membrane facing the gel. Note that many nylon membranes will only bind nucleic acids on one side. When using these membranes, care must be taken to place the DNA-binding side next to the gel. You will be using Hybond nylon membrane, which is 2-sided, so this will not be a problem. In this lab, we will be using only the liquid in the gel for the transfer. To get a more complete transfer, the sandwich may be suspended above a tank of neutralization buffer with a paper wick connecting the tank and the sandwich. This allows for a longer transfer time, and a more complete transfer. (See figures in “Southern blot” handout.)
After the transfer is complete, the membrane should be labelled to indicate when it was prepared and which side contains the DNA. Optionally, the membrane may be rinsed in 0.4 N NaOH for 1 minute to ensure that the DNA is completely denatured.
If nitrocellulose is being used, the membrane must then be baked for 2 hours at 80°C to fix the DNA to the filter. This is not necessary for nylon, which forms covalent bonds with nucleic acids under alkaline conditions.
Optionally, the gel may be tested for complete transfer of the DNA by staining in 0.5 µg/ml ethidium bromide and viewing under UV light.
Materials (per 2 groups)
1 pre-run agarose gel containing:
1 kb molecular weight markers (X 2)
0.5 µg P. ochraceus genomic DNA digested with BamHI (X 2)
0.5 µg P. ochraceus genomic DNA digested with EcoRI (X 2)
0.25 N HCl
ddH2O (double distilled H2O)
0.4 N NaOH, 0.6 M NaCl
1.5 M NaCl, 0.5 M Tris-Cl (pH 7.5)
10 X SSC
20 X SSC: 3 M NaCl
0.3 M sodium citrate · 2 H2O, pH 7.0
Hybond N+ membrane
saran wrap
Whatman 3MM filter paper
paper towels
glass plate
2 X SSC, 0.2 M Tris-Cl (pH 7.5)
Methods
Day 1:
1. Photograph your gel. (Wear a glove on the hand in contact with the gel.) A copy of the photo – with markers and sample lanes labelled – should be put in your lab book.
2. Cut gel in half between the 2 sets of lanes. Each group will use ˝ of a gel.
3. Trim off excess agarose. (The 1 kb ladder lane should also be removed.) Discard gel fragments in ethidium bromide waste.
4. Soak gel in 0.25 N HCl until bromophenol blue dye turns yellow. (Maximum 10 minutes.) Carefully empty HCl solution into sink, and rinse gel with ddH2O. Avoid splashing HCl or NaOH solutions on skin or clothes.
5. Soak gel in 0.4 N NaOH, 0.6 M NaCl for 15 minutes. Discard solution in sink, and repeat with a second 15 minute alkaline rinse. Rinse gel with ddH2O.
6. While the gel is soaking in the alkaline rinse, cut a piece of Hybond membrane to the exact size of your gel. Do not touch the membrane with your fingers – handle with gloves and/or forceps, and keep the membrane between the liner sheets until it is ready to be wetted. This protects the membrane from skin oils (which would interfere with proper wetting), nuclease contamination, and dust.
7. Float the membrane on top of a Petri dish of ddH2O until it has “wetted” by capillary action. If membrane is not evenly wetting, consult with TAs – you may need to use another piece of membrane.
8. After the gel has finished its alkaline rinse, soak the gel in 1.5 M NaCl, 0.5 M Tris-Cl (pH 7.5) for 15-30 minutes to neutralize the gel.
9. Soak the membrane in a 10 X SSC solution for 15 minutes.
10. Cut 2 pieces Whatman 3 MM paper to be exactly the same size as your gel. Handle with gloves/forceps.
11. When the gel and membrane have soaked long enough, assemble the sandwich in the order (from the bottom) saran wrap - gel - membrane - 2 sheets Whatman 3 MM paper - ~ 10-20 thicknesses folded paper towels - glass plate - weight. (To see a diagram of this, check out the “Southern blot” references on reserve in the library.) Do not move the membrane once it has been placed on the gel. Air bubbles between layers should be removed by gently rolling a 5 ml pipette across the top layer before adding the next layer of the sandwich.
12. Allow the transfer to proceed overnight.
Day 2:
1. Disassemble the blot. (Use gloves.) Before removing the membrane from the gel, note the position of the wells with a pencil, and label the membrane with your group number and day. This will allow the identification of your blot, and also allow the identification of which side of the membrane has DNA attached (the side closest to the gel.) The gel should be disposed of in ethidium bromide waste, and the paper can go in the garbage
2. Rinse membrane in 2 X SSC, 0.2 M Tris-Cl (pH 7.5) for 1 minute.
3. Place the membrane (DNA side up) on a clean, dry, piece of filter paper and let dry at room temperature. Place the membrane within a folded, labelled paper towel and leave until the next lab period.
Notes:
Plaque lifts are a technique similar to the Southern blot. It allows screening of a phage library by adsorbing phage particles onto nitrocellulose or nylon membranes. The phages and the DNA they contain are denatured in NaOH, and the denatured DNA binds to the membrane, where it can be probed to find specific phage clones containing the DNA sequence of interest. Note that many nylon membranes will only bind nucleic acids on one side. When using these membranes, care must be taken to place the DNA-binding side next to the agarose. You will be using Hybond nylon membrane, which is 2-sided, so this will not be a problem.
The plates for plaque lifts are prepared in a similar manner to those used for phage titration, with the exception that agarose is used in the overlay instead of agar. (This is because agar tends to stick to the nylon membrane.) The plates used should be several days old, or stored at 4°C for at least an hour before use, to ensure that the overlay is firmly bonded to the agar plate. Multiple lifts can be prepared from a single plate, to allow controls for false positives or tests with multiple probes.
The first step in screening a phage library is to titre the stock to find the concentration of phage particles present. This was performed in your first lab – the phage plates you will be provided with should have several thousand plaques per plate, based on titration results reported from the first lab.
This lab is equivalent to a “first screen” for a desired clone. Phages are plated at a very high concentration, to minimize the number of plates needed to find the desired clone. Because of the crowded plaques that result, it is difficult to obtain a pure culture of the desired clone. This is solved by using a “second screen”. The positive plaque or plaques from the first screen is removed from the agar with a Pasteur pipette, and used to prepare a fresh phage culture. This culture is titred, and then plated on screening plates at a lower density than the first screen. This allows pure cultures of the desired clone(s) to be isolated. A third screen may be done on the pure culture, to confirm that no contamination is present.
Materials (per group)
1 phage plate
(LB plate with 2.5 ml 0.6 % top agarose overlay inoculated with E. coli C600 and lambda CH4 containing P. ochraceus EcoRI genomic library)
Hybond N+ membrane circles
Whatman 3MM filter paper
0.5 N NaOH
1.0 M Tris-Cl (pH 7.5)
petri plates
1. Each group is provided with a prepared phage plate. Label the plate with your group number and day.
2. Preparation of membrane. (Handle with gloves and/or forceps.) Take a Petri plate-sized membrane circle. Carefully label one side of the filter with group name and day in pencil.
3. There are 2 ways that membranes can be marked for alignment with a phage plate. Either one may be used.
The first way is to cut 3 asymmetric notches on the edges of the filter before it is placed on the plate. Once the membrane has been placed on the plate, the location of the membrane can be noted by outlining the notches on the bottom of the plate.
The second method is done after the membrane has been placed on the plate. A syringe needle is used to punch sets of 1, 2, and 3 holes at different locations on the plate. (The needle should be pushed through both the membrane and the agarose, so there are identical marks on each.)
4. Using flat-tipped forceps, carefully place the membrane on top of the phage overlay pencil side down. Do not move the membrane once it has come into contact with the plate. Leave the membrane on the plate for 2-3 minutes. It should gradually wet and flatten on the agarose surface on its own.
5. Remove membrane from plate and place DNA side (pencil side) up on 2 pieces of Whatman 3MM paper soaked in 0.5 N NaOH. (This can be conveniently done in a large Petri plate.) Allow membrane to soak for 1-2 minutes (until evenly wetted) before blotting on a clean, dry piece of filter paper.
6. Wrap plate in parafilm and store in refrigerator until blots have been probed. Positive clones will be identified by comparing X-ray films with your stored plates.
7. Repeat step 5 with fresh pieces of Whatman 3MM paper.
8. Prepare 2 more Petri plates containing 1.0 M Tris-Cl (pH 7.5). Float the membrane DNA side up in the first plate for 2 minutes, and then blot dry on clean filter paper. (Make sure membrane is wetting evenly.)
9. Repeat step 8 in second plate of neutralization solution.
10. Leave membrane to dry at room temperature. Store between clean sheets of filter paper until next lab.