DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols©

Southern blotting

(last modified December 30, 2002)

Protocol

for the digestion, agarose gel-electrophoresis and Southern blotting of total genomic DNA (kindly provided by Bert Bakker and Els Voorhoeve)

Target group

The technicians, students and guests working at the clinical DNA-diagnostic department.

Responsibilities

The technicians, students and guests using digestion, agarose gel electrophoresis and Southern Blotting for DNA-diagnostic analysis of specific diseases are responsible to perform these procedures according to this protocol.

Definitions

Description

The procedure starts with the restriction enzyme digestion of genomic DNA, followed by agarose gel electrophoresis and Southern Blotting. The DNA used in this procedure is isolated according to protocol DNA isolation from blood (ALG001) or DNA isolation from chorionic villi (ALG002). The concentration of the DNA is measured according to protocol ALG003. Five µg of each DNA-sample is digested by one or more restriction enzymes. After incubation all samples are tested for complete digestion. If the samples are digested completely, the procedure can be continued, otherwise an extra incubation step is performed.

The procedure continues with the separation of the DNA fragments on an agarose gel. An electric field is applied across the agarose gel on which the DNA samples are loaded. At neutral pH the DNA-fragments have a negative charge and will therefore migrate towards the anode (positive charge). The DNA-fragments are separated according to their size; the smaller fragments will migrate faster through the gel than the larger fragments.

After separation the DNA-fragments are transferred to a nylon membrane (Southern blotting). The DNA in the gel is denatured with alkali-solution, subsequent capillary transport of the DNA fragments takes place from the gel to the nylon membrane, using the same alkali-solution. The relative positions of the DNA-fragments are preserved during their transfer to the nylon membrane.

After alkali-blotting the DNA-fragments are bound to the membrane and hybridization with a radio-labelled probe (protocol ALG005) is used to detect specific fragments.

Durables

NOTE: equivalent durables may be used

Consumables

NOTE: equivalent consumables may be used

Chemicals

NOTE: equivalent consumables may be used

Solutions

The solutions are prepared according to the protocols and autoclaved when necessary. For the solutions marked with a * a special form (with the protocol on it) is filled out. The other solutions are ready to use or made according to the protocols described here.

 

DNA isolation protocol

  1. Digestion of DNA sample
    This part of the protocol is carried out in a pre-PCR lab

    Make a pipetting-scheme on theWTEST-form using the concentration measurement of ALG 003. Complete the form before you start. The protocol below is based on a digestion of 5 µg of genomic DNA, which is sufficient for one lane on the blotgel. If necessary it is possible to take a multiple amount of DNA. The complete procedure takes 3 days. For pipetting water, spermidine and buffer there is no need to change tips
    NOTE: DNA isolated from chorionic villi (ALG002) contains a lot of RNA. This influences the measured DNA concentration (ALG003). To get equal signals on your blot compared to normal blood-derived DNA samples, take twice the amount for the digestion (if possible !)
    1. based on the measured DNA-concentration (ALG003), calculate which volume has to be pipetted from each DNA sample and write down this volume on the WTEST-form
    2. write down the amount of water (in µl) you have to add, to get a total volume of 23 µl
      NOTE: in some cases it is necessary to adjust this volume
    3. mark the 1.5 ml Eppendorf safe lock tubes with the complete DNA number and the code(s) of the enzyme(s) used
    4. prepare a mix sufficient for all digestions, containing 3 µl of the 10 x restriction enzyme reaction buffer mentioned in the disease specific protocol and 1 µl 100 mM spermidine
      NOTE: in some cases it is necessary to adjust the amount of 10 x restriction enzyme reaction buffer used, note this on the WTEST-form
    5. first pipet the amount of water in each tube, then add 4 µl of the restriction enzyme reaction buffer/spermidine mix
    6. using Cell Saver tips, add the calculated volume of each DNA sample
      NOTE: use a new tip for every DNA sample. The total volume is 27 µl, or a multiple thereof
    7. incubate 30 min in the 65oC incubator, cool down to room temperature and add 15 units restriction enzyme (3 units for each µg of DNA)
      NOTE: never add more enzyme than 10 % of the total volume because glycerol inhibits enzyme activity. Write down the amount of enzyme used on the WTEST-form
    8. mix carefully and spin down 5-10 sec in the Eppendorf centrifuge
    9. incubate overnight in the 37oC incubator
      NOTE: for some diseases enzymes with different incubation temperatures are used, or double digestions are performed (ee the disease specific protocol)
  2. Analysis of the digestion
    This part of the protocol is partly carried out in a pre-PCR lab (preparation of the samples) and partly in a post-PCR lab (preparation of the gel and electrophoresis of the samples). Depending on the capacity needed, two gel sizes are used: a 15 x 45 cm gel (capacity 6 x 22 lanes) or a 15 x 10 cm gel (capacity 2 x 22 lanes)
    1. preparation of a 15 x 45 cm [or 15 x 10 cm] gel
      The Sub-Cell GT Agarose Gel Electrophoresis System is used and the gel is casted using a Gel Caster. NOTE: to insure the long life of Sub-Cell GT parts, never use ethanol or other agressive chemicals for cleaning.
      1. prepare a 0.8 % agarose testgel in TBE-buffer. For a 15 x 45 cm gel add 2.4 g agarose to 300 ml TBE-buffer in an erlenmeyer and mix. [for a 15 x 10 cm gel add 1.2 g agarose to 150 ml 1 x TBE-buffer]
      2. heat the mixture in the microwave oven until the agarose is dissolved and the solution is boiling. Shake the mixture gently during the heating process to homogenise
      3. add Elga water to a final volume of 300 ml [or 150 ml] and add 6 µl EtBr (10 mg/ml) [or 3 µl] and mix by gentle shaking
      4. rinse the gel tray and the rubber parts of the Gel Caster with Elga water and dry them with a paper towel. Disengage and slide the movable wall to the open end of the Gel Caster by turning and lifting the cam lever upward. Place the open end of the gel tray against the fixed wall of the Gel Caster. Slide the movable wall against the edge of the gel tray. To seal the open tray ends, engage the cam lever by turning and pressing downward simultaneously, until resistance is felt
      5. the agarose should be allowed to cool to approximately 60oC before pouring into the gel tray (cool the erlenmeyer under cold running tap water). Pour the melted agarose into the tray and place the combs. Avoid air bubbles. Wait 30-60 min until the gel has solidified
      6. carefully remove the combs from the solidified gel by lifting them straight up
      7. disengage the cam lever by turning and lifting upward. Slide the movable wall away from the gel tray. Remove the gel tray from the Gel Caster and place it onto the Sub-Cell base
      8. submerge the gel beneath 2 to 6 mm TBE/EtBr-buffer
    2. preparation and electrophoresis of the samples
      1. add 1.5 µl of the digested DNA to 10 µl of 2xSLM in a micro titre dish
      2. load the samples on the gel and run for 1-2 hours at 100-200 Volt
        NOTE: the samples migrate towards the positively charged anode
      3. print a photo of the gel with the Imager (use the print button on the computer so that date and time will be printed on the photo)
        NOTE: since it is UV transparent, it is not necessary to remove the gel from the gel tray
      4. put the photo on the WTEST-form, mark the slots and write down the DNA numbers. Look critically at the photo to see whether all digestions are complete (see Troubleshooting). Write on the WTEST-form all the additional steps performed
        NOTE: store the digested samples at 4oC in the refrigerator at the pre-PCR lab or cold room. If not used on day 2, storage should be at -20oC in the freezer in the pre-PCR lab
  3. Agarose gel electrophoresis
    this part of the protocol is partly carried out at a pre-PCR lab (preparation of the samples) and partly at the post-PCR lab (preparation of the gel, electrophoresis of the samples)
    1. preparation of a 20 x 25 cm blot gel
      the Owl Scientific electrophoresis system is used and a gel is casted in a casting tray
      1. prepare a 0.7 % agarose blotgel in TAE-buffer. Add 2.45 g agarose to 350 ml TAE-buffer in an erlenmeyer and mix
        NOTE: a higher (0.8 %) or lower (0.5 % or 0.6 %) percentage of agarose can be used according to the disease specific protocol
      2. heat the mixture in the microwave oven until the agarose is dissolved and the solution is boiling
        NOTE: shake the mixture gently during the heating process to homogenise
      3. add Elga water to 350 ml and 14 µl EtBr (10 mg/ml), mix by gentle shaking
      4. slide the gasketed end gates into the outermost grooves on either side of the tray (the end gates should be inserted with the gasket side facing out
      5. the agarose should be allowed to cool to approximately 60oC before pouring into the tray (to cool, keep the erlenmeyer under cold running tap water). Avoiding air bubbles, pour the melted agarose into the tray
      6. once the agarose has been poured, insert one comb (24 or 28 wells) in the groove, approximately 2 cm from the top of the gel
      7. when the gel has solidified (after 30-45 min), lift the end gates out of the chamber
      8. place the gel tray in the buffer chamber filled with TAE/EtBr-buffer, carefully remove the comb
        NOTE: to avoid damaging the wells, lift the comb straight up
    2. preparation and electrophoresis of the samples
      1. heat the 10 x SLM by keeping the tube under running hot tap water to make it completely fluid. Mix the digested DNA samples (about 25 µl) with 3 µl 10 x SLM
      2. load in each slot 25 µl of the sample. Write down the order in which the samples are loaded on the WBLOT-form. Load a maximum of 6 samples next to each other and skip one slot. Electrophoresis is overnight at 30-50 Volt
        NOTE: when specified in the disease specific protocol, a two times overnight electrophoresis is performed
  4. Southern blotting
    This part of the protocol has to be carried out at a post-PCR lab
    NOTE: alkali-solution 1 is used to transfer the DNA in 4 to 6 h, alkali-solution 2 is used for overnight blotting
    1. for every blot, prepare a fresh alkali-solution
    2. clean all the glass plates and trays with water and, if necessary, with "Jif"
    3. put the gel on the UV transilluminator
      NOTE: clean the UV transilluminator with water before and after putting the gel on the transilluminator
    4. cut the gel to the correct size (just above the slots, and just around the DNA)
    5. make a print-out of the gel with the Imager system (use the print button on the computer, date and time will be printed)
    6. stick the print-out on the WBLOT-form
    7. turn the gel upside down between two glass plates
    8. denature the gel two times 15 min in the alkali-solution under gentle agitation (number the gels when more then one gel is used in the blotting procedure). Refresh the solution after 15 min
      NOTE: in the mean time prepare everything needed for blotting
    9. cut the following to size
      • two GB002 papers (35 x 19 cm or 46 x 19 cm)
      • one GB002 paper (20 x 19 cm or 29 x 19 cm)
      • one Hybond XL membrane, 1 cm larger than the gel. Mark the membrane with a black pen with the date and number of the gel (if more then one gel is blotted). Put Elga water in a tray, soak the membrane in it, pour off the Elga water and leave the membrane in alkali-solution for at least 5 min
      • one GB002 paper same size as the gel
      • one GB003 paper same size as the gel
    10. start to build the "blotting tower"; put a glass plate on top of 2 plastic trays (20 x 16 cm) containing alkali-solution
    11. soak the two GB002 papers (35 x 19 cm or 46 x 19 cm) in alkali-solution and put them on the glass plate. Remove air bubbles by rolling with a glass pipet
      NOTE: repeat this step every time you put a new layer on the blotting tower
    12. soak the GB002 paper (20 x 19 cm or 29 x 19 cm) in the alkali-solution and put it on the other papers
    13. add alkali-solution to soak the paper bridge completely
    14. put the denatured gel, still upside down, on a glass plate and carefully transfer it to the GB002 paper bridge
      NOTE: be extra careful with a 0.5 % gel
    15. put slices of film around and just under the edges of the gel
      NOTE: this ensures a capillair flow through the gel to the membrane
    16. add alkali-solution to soak the gel completely
    17. with the marked corner in the correct position, put the membrane on the gel
      NOTE: see the disease specific protocols for the exact positions of the marks
    18. soak the GB002 and the GB003 papers (with the same size as the gel) together in alkali-solution and put them on the membrane
    19. fold paper towels in about the same size as the gel. Make a pile of about 10 cm high and put this on the blotting tower
    20. place a glass plate together with a weight of about 250 gram on top
      NOTE: do not use a weight when a 0.5 % agarose gel is blotted
    21. blotting takes place in 4 to 6 h (with alkali-solution 1) or overnight (with alkali-solution 2)
    22. after blotting remove the weight, glass plate, paper towels and papers. Neutralize the membrane in neutralisation buffer for 1-5 min and dry it between filter paper
    23. check the membrane for correct blotting and possible introduction of air bubbles using a UV transilluminator (cover it with Saran Wrap to protect the membrane). Mark all empty lanes and if possible the slots on the DNA side of the membrane. Check this with the print-out of the blotgel before cutting the membrane
    24. cut the membrane to the correct size for hybridization (see ALG005) and write, with a black pen on the DNA side, on each piece the WBLOT number, the enzyme(s) used and the DNA numbers
    25. membranes are now ready for hybridisation according to ALG005
      NOTE: store the dried membranes at RT in envelopes on WBLOT number
    26. throw away remaining alkali solutions, the blotted gel and all paper used in the procedure. Rinse the glass plates and trays with water to remove all alkali solution. Clean the table with water

References

[1] Maniatis, Sambrook, Fritsch: Molecular Cloning, 2nd edition 9.31

Troubleshooting

  1. when some of the samples are not completely digested, add more enzyme (10-20 units) and incubate again for 3-4 hours at the appropriate temperature. The addition of 1 µl 100 mM spermidine can also make a difference. If problems remain unsolved perform "Troubleshooting 2"
  2. when digestions of a certain DNA sample are all incomplete or remain incomplete, it might be useful to perform an extra purification step according to ALG 026. This is necessary to get rid of proteins or other contaminants disturbing the digestion.

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