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Method: Making Combination Markers for Size Standards on Southern Blots

December 5, 1990

Matthew S. Holt


Preface:

Time required:

Procedure:

  1. Digest 50 g lambda DNA (BRL) in three separate digests using 150 units each of BglII, BstEII, and XhoI in a total volume of 400 l. Incubate at the appropriate temperature for 4-6 hours (DNA concentration 125 ng/l). Note: BstEII digests should be done at 60 degrees C.
  2. Add another 150 U enzyme (in a 10 l volume at 1X the correct restriction enzyme buffer) to each digest. Incubate for another 4-6 hours or overnight.
  3. Remove a total of 4 tests from the digests: 4 l (~500 ng) of each digest for the first three tests, and 1.4 l of each digest pooled for the fourth (~ 500 ng). Add 10 l of 1X glycerol stop mix to each and run on a mini-gel along with uncut lambda DNA concentration standards. Run the gel until the bromophenol blue dye is 2-3 inches from the well tooptimize the fragment separation.
  4. Stain and photograph the test gel. Check for partials and verify fragment sizes. Check DNA concentrations against the lambda standards.
  5. Add another 150 U enzyme (in a 10 l cocktail) to any digest with partials and incubate for 4-6 hours. Test a sample for complete digestion on a minigel. Once the digests are complete, store in -20 degrees C freezer until phenol-chloroform extraction.
  6. Phenol extract each digest by adding 400 l of phenol pH 8.0 to each tube, vortex to mix, and spin at 12,000 rpm for 5 minutes. Remove the aqueous phase (on top) and transfer to a clean labeled tube.
  7. Chloroform extract each digest by adding 400 l chloroform to each tube, vortex to mix, and spin at 12,000 rpm for 5 minutes. Remove the aqueous phase and transfer to clean labeled tube.
  8. Precipitate DNA by adding 45 l 3M NaAcetate pH 5.2 and 900 l -20 degrees C 100% EtOH to each tube. Spin immediately in a microfuge at 12,000 rpm at 4 degrees C for 30 minutes. Decantand rinse each pellet with 400 l -20 degrees C 70% EtOH, spin as above for 5 minutes, decant and dry the pellet.
  9. Resuspend each DNA pellet in 50 l 1X TE (DNA concentration 1g/l).
  10. Prepare a full size 0.8% TA gel for another visible DNA test. The purpose of this gel is to equilibrate the three individual digest concentrations so that you can accurately dispense 2 g of each: Dilute 2 l (~2 g) of each digest with 18 l of 1X TE (100 ng/l). Prepare four tests: 5 l of each diluted digest (~500 ng) for the first three, and 1.7 l of each diluted digest pooled for the fourth. Add 10 l of 1X glycerol stop mix to each test sample. The remainder of these dilutions can be stored at -20 degrees C and used as visible markers. Run the test samples on the gel along with uncut lambda concentration standards of 300, 400, 500, and 600 ng. Run the gel at 80 V for 6 hours, stain and photograph. If necessary, repeat step 10 after adjusting the DNA volumes. Take accurate notes of any adjustments.
  11. Prepare another full size 0.8% TA gel for a Southern transfer.
  12. Make a mixture of the three digests that contains 6 g total DNA (2 g of each) in a total volume of 10 l 1X TE. Unless adjustments have been made, this equals 2 l of each digest plus 4 l of 1X TE.
  13. Add 2 l 0.5 M EDTA pH 8.0 to the mixture (this is the 500 ng/l stock).
  14. Dilute 2 l ( 1 g) of the mixture in 1 ml of 1X TE (this is the 1 ng/l stock).
  15. Remove 10 l of this dilution (10 ng) and mix with 990 l of 1X glycerol stop mix (final DNA concentration is 10 pg/l). Ideally, 15 l of this mix should equal 150 pg of DNA.
  16. Heat to 50C for 10 minutes prior to loading to melt cohesive ends. Load the gel with 5, 10, 15, 20, and 25 l of the final mix (along with previous finished markers if available for a control lane). Run the gel at 50 V for 20 hours.
  17. Transfer gel and prepare the blot as per Southern Transfer protocol.
  18. Incubate the blot in 10 ml of pre-hyb at 50C for 1 hour. Add 1x107 dpm of lambda probe (in 2 ml of warm pre-hyb) and hybridize for 4 hours to overnight on hybing rocker.
  19. Wash twice at 65C in 200 ml 2X SSC for 15 minutes each. Wash once at 65 degrees C in 500 ml0.1X SSC and 0.5% SDS.
  20. Monitor the blot and continue washing in fresh 0.1X SSC and 0.5% SDS until the backround is low (i.e. no signal on blot edges) but signal from markers is strong - 0.1 - 0.3 mRem/hr. Expose a film to the blot for 8 - 12 hours at room temperature (no intensifying screen).
  21. Develop the film and verify fragments. The purpose of this hybridization is to determine if the fragment ratios are equal. Repeat gel if the fragment ratios are unequal. If fragment ratios look acceptable strip blot and rehybridize in a bag with other blots. Wash as usual and expose as usual (3 days).
  22. Develop film and determine which loading amount (5, 10, 15, 20, 25 l) is best and adjust the DNA concentration so that 15 l of the finished markers will give the best signal (e.g., if markers are too dilute, prepare another dilution of the 1 ng/l stock by diluting 15 l (instead of 10 l) with 1 ml 1X glycerol stop mix).
  23. Store as:

Lambda molecular weight size markers ("Lambda combo markers"):

* indicates lambda end fragments. When using the lambda size standard, be sure to 'melt' the cohesive ends with a 5 minute heat treatment (50 degrees C) before loading the gel. Otherwise these fragments may not be visible and different combinations (visible if not melted) of the cohesive end fragments will interfere with the interpretation.