Gel-purified, agarased YACs for yeast transformation
Protocol from Andreas Gnirke (formerly of the Olson lab), with modifications by Anja van Brabant (Fangman/Brewer lab)
1. Yeast DNA plugs at 4 X 109 cells per ml (2 X 109 cells per 0.5 ml plug). See plug-making protocol.
2. SeaPlaque GTG agarose (FMC, Rockland, ME, Cat. No. 50112)
3. Modified agarase buffer (10 mM BisTris-HCl, pH 6.5, 1mM EDTA, 100 mM NaCl). Make 10X concentrated stock solution, autoclave and store in aliquots at -20oC.
4. Agarase (New England Biolabs Cat. No. 392L, recombinant, 1u/µl).
5. Cut-off blue and yellow tips, autoclaved.
6. Microcentrifuge concentration cartridges (Millipore Cat. No. UFC3TTK25, 30000 MWCO).
1. CHEF gel (1% low-melt SeaPlaque agarose, 0.5X TBE, ca. 0.7cm thick). Load 4 X 2/3 of a plug per gel. Use same switching time as with regular agarose, but run 1.5X as long (e.g., for 600 kb YAC: 36h/160V/50s).
2. Stain edges of gel.
3. Cut out non-stained YAC band. Try to cut out a gel slice (ca. 3-4 mm) that is thinner than the YAC band itself. Cut gel slice in 1 cm pieces (<0.5 g) and put each piece in a separate 1.5 ml Eppendorf tube. Stain rest of gel and confirm that the correct band was excised.
4. Determine the weight of agarose. Add 2 vol. (1 g=1 ml) 1 X agarase buffer (100 mM NaCl). Incubate 1 h at room temperature. Invert from time to time. Replace buffer with 2 vol. of fresh buffer and continue equilibration for another hour.
5. Remove buffer completely. Spin agarose at very bottom of tube (a few seconds in microcentrifuge). This step is not important but avoids subsequent problems with air bubbles.
6. Incubate 10 min at 68oC. Do not invert to check if melted.
7. Incubate 5 min at 40oC.
8. Add agarase (1 µl per 100 µl gel). Mixing in the agarase is the critical step of the protocol.
- Method 1: add agarase (e.g. 4 µl). To mix, slowly and gently draw solution into cut-off blue tip and pipette back into tube. (Once is sufficient).
Method 2: Gently stir melted agarose with yellow tip while adding agarase.
9. Incubate 2 h at 40oC.
10. Clear by centrifugation (15 min in microcentrifuge at room temperature).
11. Transfer ca. 80% of supernatant into fresh tube. Use cut-off tip.
** I don't do step #12 because I want to keep all of the DNA for the transformation.
12. Remove 30-40 µl for analytical CHEF gel. Use 5 µl to determine concentration on minigel (use lambda DNA 1, 2, 4, ...128 ng/lane as marker). Store rest at 4oC. Concentration should be ca. 0.5-1ng/µl.
** I always concentrate the DNA to minimize the volume used in the transformation. Also, I combine the contents of two tubes into one concentration cartridge.
13. DNA can be further concentrated using Millipore microcentrifuge concentration cartridges: load cartridge with up to 400 µl agarased DNA.
14. Spin at <5000 x g in microcentrifuge at RT.
15. Check volume of filtrate after a few minutes. Continue until 80-90% of initial volume has passed the membrane. I concentrate until approximately 50-100 µl remains. (Time will depend on centrifuge. In our centrifuge--6500 rpm, 2900 x g--it usually takes 6-8 minutes.)
16. Let sit for 1h at RT.
17. Carefully "resuspend" DNA with cut-off yellow tip (3X in and out is fine.)
** Again, I skip step #18.
18. Determine concentration and analyze on pulsed-field gel.
19. Use all of one tube (50-100 µl) for LiAC transformation, always handling the DNA very gently and using cut-off pipette tips. Plate on selective plates. Colonies may come up on the plates more slowly than the positive control. Restreak any colonies that appear and confirm the presence of the full-length YAC by making plugs from these transformants and running a CHEF gel. It's also useful if the recipient strain has a different karyotype since this can be used to confirm that the YAC has indeed been moved to a new strain.
1. It is important to use concentrated yeast DNA plugs.
2. Andi Gnirke tried large-scale preps in one tube (ca. 5 ml agarose melted in 15 ml tubes, mixing by inverting the tube), and it did not work. It is safer to cut a big gel slice into small pieces (<0.5 ml) and do several small-scale preps.
3. The melted agarose should be treated as gently as possible (i.e., use cut-off pipette tips, pipette slowly, etc.)
4. This protocol doesn't use polyamines. Gnirke found that the presence of 100 mM NaCl protects the DNA from being sheared (compared to 0 or 30 mM NaCl). Addition of 0.75 mM spermidine and 0.3 mM spermine at 100 mM NaCl appears to have no effect. At lower NaCl concentration (e.g., 30 mM) polyamines protect the DNA (but not necessarily better than 100 mM NaCl without polyamines). At low NaCl concentrations, polyamines can precipitate the DNA, and one usually loses all the DNA during a short spin in a microcentrifuge (sometimes the DNA seems to vanish even at 1 x g overnight in the refrigerator.)
Questions? email Anja
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