Method: Making Combination Markers for Size Standards on Southern Blots

December 5, 1990

Matthew S. Holt

Preface:

Digesting lambda DNA (BRL) with a specific restriction enzyme will result in lambda DNA fragments of known size. Three separate lambda digests (BglII, BstEII, and XhoI) will give 23 fragments with a molecular weight range of 60 bp to 33,498 bp (see illustration below). The three digests are pooled, diluted, and used as molecular weight size standards for Southern transfers.

Optimal marker lanes signal on autoradiographs is achieved by loading 150 pg of markers per lane (this amount is not visible with ethidium bromide staining), and probing with 5 x 106 counts of labeled lambda DNA per blot up to 10 blots, or 1 x 107 counts per blot above 10 blots, for a three day -80 degrees C exposure.

Marker mixes are made and stored at 500 ng/µl and 1 ng/µl. Diluted markers ready for running on the Southern gel are made in batches by mixing approximately 100 µl of the 1 ng/µl dilution in 10 ml 1X glycerol stop mix.

NOTE: Markers should be heated to 50 degrees C for 10 minutes prior to loading to melt the lambda cohesive ends.

Time required:

one week

Procedure:

1. Digest 50 µg lambda DNA (BRL) in three separate digests using 150 units each of BglII, BstEII, and XhoI in a total volume of 400 µl. Incubate at the appropriate temperature for 4-6 hours (DNA concentration ‰125 ng/µl). Note: BstEII digests should be done at 60 degrees C.
2. Add another 150 U enzyme (in a 10 µl 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 µl (~500 ng) of each digest for the first three tests, and 1.4 µl of each digest pooled for the fourth (~ 500 ng). Add 10 µl 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 µl 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 µl 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 µl 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 µl 3M NaAcetate pH 5.2 and 900 µl -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 µl -20 degrees C 70% EtOH, spin as above for 5 minutes, decant and dry the pellet.
9. Resuspend each DNA pellet in 50 µl 1X TE (DNA concentration ‰ 1µg/µl).
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 µg of each: Dilute 2 µl (~2 µg) of each digest with 18 µl of 1X TE (100 ng/µl). Prepare four tests: 5 µl of each diluted digest (~500 ng) for the first three, and 1.7 µl of each diluted digest pooled for the fourth. Add 10 µl 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 µg total DNA (2 µg of each) in a total volume of 10 µl 1X TE. Unless adjustments have been made, this equals 2 µl of each digest plus 4 µl of 1X TE.
13. Add 2 µl 0.5 M EDTA pH 8.0 to the mixture (this is the 500 ng/µl stock).
14. Dilute 2 µl (‰ 1 µg) of the mixture in 1 ml of 1X TE (this is the 1 ng/µl stock).
15. Remove 10 µl of this dilution (‰10 ng) and mix with 990 µl of 1X glycerol stop mix (final DNA concentration is 10 pg/µl). Ideally, 15 µl of this mix should equal 150 pg of DNA.
16. Heat to 50šC for 10 minutes prior to loading to melt cohesive ends. Load the gel with 5, 10, 15, 20, and 25 µl 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 50šC 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 65šC 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 µl) is best and adjust the DNA concentration so that 15 µl of the finished markers will give the best signal (e.g., if markers are too dilute, prepare another dilution of the 1 ng/µl stock by diluting 15 µl (instead of 10 µl) with 1 ml 1X glycerol stop mix).
23. Store as:
    • 1 µg/µl individual digested lambda fragments at -20 degrees C.
    • 500 ng/µl of pooled DNA fragments at -20 degrees C.
    • 1 ng/µl dilution of pooled DNA fragments at -20 degrees C.
    • 10 pg/µl dilution of pooled DNA fragments in 1X glycerol stop mix at 4 degrees C; many tubes of this dilution should stored the lab for use with the Southern gels.

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

Combined digests of BstEII, BglII, and XhoI digests of wild type lambda DNA.

The useful range in Southern blots is from 1264 to 33498 bp.

* 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.