Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis
- Zymolyase 20T (Sunrise Science Cat, San Diego, CA, cat. #NO766391)
- Proteinase K (VWR, Radnor, PA, cat. #97062-238)
- Low-melting point (LMP) agarose (Invitrogen, Carlsbad, CA, Cat #16520)
- N-Lauroylsarcosine sodium salt (Sigma-Aldrich, St-Louis, MO, cat. #L5125)
- EDTA (Thermo Fisher Scientific, Inc. Hampton, NH, cat. #BP-120)
- Tris-HCl, pH 7.4 (Lonza, Conley, GA, cat. #51237)
- Pulse Field Certified Agarose (Bio-Rad, Hercules, CA, cat. #1620137)
- Ethidium bromide, 10 mg/ml (Teknova. Inc. Hollister, CA, cat. #E3050)
- Tris Base (Thermo Fisher Scientific, Inc., cat. #BP152-1)
- Boric Acid (Thermo Fisher Scientific, Inc., cat. #A74-1)
- T10E50 (10 mM Tris-HCl, pH 7.4, 50 mM EDTA): Mix 10 ml of 1 M Tris-HCl, pH7.4 with 100 ml 0.5 M EDTA in 890 ml dH2O.
- T10E500 (10 mM Tris-HCl, pH 7.4, 500 mM EDTA): Mix 1 ml of 1 M Tris-HCl, pH7.4 with 99 ml 0.5 M EDTA.
- 1% LMP agarose: Mix 25 ml 0.5 M EDTA with 75 ml dH2O, add 1 g of low-melting point agarose and heat until agarose is completely dissolved and in solution.
- Zymolyase stock solution (20 mg/ml): Add 20 mg to 1 ml 50% glycerol. Store at −20°C.
- Zymolyase working solution: Add 30 µl of Zymolyase stock solution (20 mg/ml) to 3 ml of T10E50 (this is enough for 30 wells and can be adjusted accordingly). Make working digestion buffer immediately prior to use, discard unused portion.
- Sarkosyl stock solution: Dissolve 1 g of N-Lauroylsarcosine sodium salt in 100 ml T10E500.
- Proteinase K stock solution (20 mg/ml): Add 20 mg proteinase K to 1 ml 50% glycerol. Store at −20°C.
- Proteinase K/sarkosyl working solution: Add 30 µl of proteinase K (20 mg/ml) stock solution to 3 ml of sarkosyl stock solution (this is enough for 30 wells and can be adjusted accordingly), make immediately prior to use and discard unused portion.
- 10× TBE: Add 108 g Tris Base, 55 g boric acid, 40 ml 0.5 M EDTA to a final volume of 1 L dH2O.
- 0.5× TBE: Mix 100 ml of 10× TBE with 1900 ml of dH2O.
- Reusable 48 well plate-5 ml rectangle well, pyramid bottom clear polypropylene (E&K Scientific, Santa Clara, CA, cat. #EK-2044)
- Breathe-easy gas permeable sealing membrane (RPI Corp, Mount Prospect, IL, cat. #248738)
- 30°C Shaker/incubator (New Brunswick Scientific, Edison, NJ, Model G25)
- Centrifuge with plate rotor (e.g. Beckman, Brea, CA, Allegra 25R Centrifuge, S-5700 rotor)
- Repeater pipette, capable of delivering 100–1000 µl (e.g. Eppendorf Repeater Plus)
- Vortex with plate adaptor (Benchmark, cat. #AM10014)
- Water bath at 37°C, 55°C and 75°C
- Vacuum aspirator (2 L filter flask + vacuum tubing and pipette tip)
- Rocking platform (Fisher Scientific, Model #88861045)
- 3M® book tape or similar packing tape (3” wide)
- CHEF-DR II system (Bio-Rad)
Inoculation and growth • TIME 10 min
1.Add 1 ml of liquid growth medium to each well of an E&K Scientific 48 well plate.
2.For each sample, pick a small amount of cells from an isolated colony with a sterile toothpick and insert into medium-filled well. Remove toothpicks.
3.Cover plate with Breathe-easy gas permeable sealing membrane.
4.Grow cells at 30°C with shaking overnight.
5.Measure optical absorbance at A600 for all cultures. Cell amount can be varied to achieve A600 between 2 and 4.
Washing, preparation of agarose plugs and digestion of cells • TIME 35 min (excluding 4 h to overnight incubation)
6.Pellet cells from overnight growth. Centrifuge cells in the plate using a plate rotor, 5 min at 5000 rpm.
7.Remove supernatant by quickly inverting the plate, or with aspirator.
8.Wash cells by adding 1 ml of dH2O to each well.
9.Resuspend cells by mixing on vortex for ~1 min using a plate adaptor.
10.Centrifuge plate for 5 min at 5000 rpm at room temperature.
11.Remove supernatant by briefly inverting the plate and flicking as above, or with aspirator.
12.Wash cells by adding 1 ml of T10E50 to each well then resuspend, as in steps 8 and 9.
13.Centrifuge plate for 5 min at 5000 rpm.
14.Remove supernatant by briefly inverting the plate and flicking as above, or use aspirator.
15.Prepare 1% LMP agarose by adding 0.5 g LMP agarose to 50 ml of 0.125 M EDTA and carefully melting to homogeneity on low setting in microwave.
16.Quickly add 100 µl of Zymolyase working solution per well using repeater pipette.
17.Add 150 µl of 1٪ LMP agarose per well with repeater pipette. Immediately after the addition of LMP agarose, resuspend the cells for 1 min using a vortex with plate adapter.
18.Place the plate on ice for 5 min to allow agarose to set. Agarose plugs will solidify and adhere to the bottom.
19.Add 150 µl of T10E500 per well and cover the entire plate with 3M® book tape.
20.Incubate for at least 4 h in a 37°C water bath.
Adding proteinase K • TIME 5 min (excluding overnight incubation)
21.Add 100 µl of proteinase K/sarkosyl working solution to each well.
22.Re-seal the plate with fresh 3M® book tape.
23.Incubate overnight in a 55°C water bath.
Plug washing • TIME 20 min hands-on
24.Add 1 ml of room temperature dH2O to each well/plug and immediately aspirate.
25.Wash plugs by adding 1 ml T10E50 to each well then rock gently on rocking platform for 20 min.
26.Remove T10E50 with aspirator, as above.
27.Wash plugs by adding 1 ml fresh T10E50 to each well then rock gently on rocking platform for 40 min.
28.Remove T10E50 with aspirator, as above.
29.Wash plugs by adding 1 ml fresh T10E50 to each well then rock gently on rocking platform for 60 min.
Preparation of PFG and loading plugs • TIME 15 min hands-on
30.Weigh out 1.5 g Pulsed Field Certified agarose in a 250 ml flask, and add to 150 ml of 0.5× TBE. Microwave, checking frequently for boiling, and confirm that agarose is completely melted.
31.Prepare gel frame on level surface, with comb positioned evenly. Comb teeth approximately 1 mm from gel frame base.
32.When agarose has cooled to 55°C, slowly pour into gel frame and let cool for 30 min.
33.Pour remaining 0.5× TBE into gel rig chamber and cool to 15°C.
34.From the 48-well plate, remove T10E50 from wells with aspirator, ensuring that all droplets are removed from the sides of wells.
35.Incubate the plate in a 75°C water bath for 5 min.
36.Transfer the multi-well plate from 75°C bath to 70°C heat block.
37.Clip off 20 µl pipette tip with razor blade. Tip orifice diameter should be ~1 mm. One tip can be used for loading all plugs into gel.
38.Carefully remove comb from gel.
39.Load 20 µl of each liquid agarose plug sample with a clipped-off pipette tip.
40.After all the samples are loaded, immediately remove plate from heat block, and place gel into cooled buffer.
41.Let plugs in 48-well plate solidify then overlay each plug with 250–500 µl of T10E500 for long term storage.
42.Cover plate with 3M® tape and store at 4°C, for up to 6 months.
43.Run gel at optimized parameters, for instance for Saccharomyces, 6 V, 60–100 s switch time, for 30 h at 15°C.
44.After run, remove gel and place in container with 1 L 0.5× TBE buffer for staining.
45.Add 10 µl of 10 mg/ml ethidium bromide. Mix gently on rocking platform for 30 min.
46.Visualize chromosomes on UV transilluminator.
|5||Chromosome plug not clear in step 21||
|6||Loss of cells during washing||
|16||Less DNA than expected in gel matrix||
|17, 18, 32||Smearing of DNA bands||
|7, 11, 14, 21, 23, 25, 31||Loss of plug||
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