McClean:URA Pop-out

Overview

This protocol is used for "popping" URA3 out of the genome with your desired DNA in it's place. It is basically a "normal" LiAc transformation with a few additional important details (see Notes). Selection is done on 5-FOA to select for ura- cells. This method works because the URA3 gene encodes orotidine 5´- phosphate decarboxylase (which catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, converting OMP into uridine monophosphate (UMP)). This protein also converts 5-fluoroorotic acid (5-FOA) to the toxic compound 5-fluorouracil. Therefore, URA+ cells are killed in the presence of 5-FOA while ura- cells are resistant. In the lab we usually use this for doing "clean" genetic modifications, ie when we don't want drug markers, etc "hanging around" in the genome. We "pop-in" URA3 where we want to do the modification, and then pop it out with our modified DNA of interest using this protocol. In practice, the URA3 that we pop-in usually comes off of a pRS vector.

Materials

• 100x 5-Fluoroorotic acid Solution (Zymo Research, #F9003 100mg/ml in DMSO )
• Uracil (MP Biomedicals, #103204)
• 5-FOA selection plates
• 100mM LiAC
• 50% w/v PEG MW 3350 (Sigma P3640)
• Sterile H20
• Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
• Sterile 4mm glass-beads for plating (Fisher 11-312B)

Reagents

5-FOA Plates

For 500ml:

• 5 ml 100x Zymo Research liquid 5-FOA
• 150ml 3.33x YNB w/o amino acids
• 50ml 20% glucose
• 50ml KS-URA (10x) amino acid solution
• 250ml 2x bacto agar
• 25mg uracil

Microwave the bacto agar to re-liquify. Allow to cool to the touch but not to solidify. Add the rest of the components (protocols can be found under media for the basic items such as YNB w/o amino acidS) to the bacto-agar. Swirl the bottom to mix the components well. If the agar is not mixing, microwave the media for short periods (30s) until everything mixes well.

Polyethylene glycol PEG 50% w/v (Sigma P3640)

• Make up 50% w/v with H₂O and filter-sterilize with a 0.45uM filter unit (Nalgene 295-4545 or similar). It will take a long time for the PEG to work it's way through the filter, be patient. We don't autoclave the PEG. Store in a tightly capped container to avoid evaporation.

Single-stranded carrier DNA (Sigma D1626)

1. Weight out 200mg of the DNA into 100ml of TE buffer. Disperse the Dna into solution by drawing it p and dwn repeatedly in a 10-ml pipette. Mix vigorosly on a magnetic stirrer for 2-3 hours or until fully dissolved. Alternatively, leave the covered solution mixing at this stage overnight in a cold rom.
2. Aliquot the DNA into 100μL portions and store at -20°C.
3. Prior to use, the aliquot should be boiled and then quickly cooled on ice. We use a thermocycler to heat the DNA to 95°C for 25 minutes and then rapidly cool it on ice.

Once the salmon sperm has been boiled it can be freeze-thawed 3 or 4 times before transformation efficiencies begin to decrease. In practice, we boil the DNA before every transformation.

TE Buffer (pH 8.0)

• 10 mM Tris-HCL (pH 8.0)
• 1.0 mM EDTA

1.0M Lithium acetate stock solution (LiAc)

• Prepare as a 1.0 M stock in distilled deionized H₂O; filter-sterilize. The final pH should be between 8.4 and 8.9

Glass-beads for plating (Fisher 11-312B)

• Pour beads into a small glass bottle (typically wide-mouthed 100ml or 250ml bottles work well) and autoclave on a 15 minute dry cycle to sterilize

Day 1

1. Inoculate the strain to transform from a single colony into 5mls of SC-URA in a test tube. Put on the roller drum at 30°C overnight.

Day 2

1. Inoculate 50 ml of SC-URA with 500 μL of the YPD overnight culture in a 250 ml flask. The 500 µl volume is approximate, and depends on the density of the strain you inoculate.
2. Grow in shaking incubator for about 3-5 hours.
3. Turn on 42°C water-bath (for heat-shock) if it is not already on.
4. Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min. Resuspend pellet in 25 ml of sterile water by vortexing briefly. Pellet again and then resuspend in 1 ml of 100 mM LiAc.
5. Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
6. Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down. Aliquot 50 μL into 1.5 ml tubes (1 for each transformation). Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
7. Add 300 μL T mix to each eppendorf tube of cells. Per one transformation reaction add IN ORDER:
   • 240 μL 50% PEG 3350
   • 35 μL 1.0 M lithium acetate
   • 25 μL 2 mg/ml sssDNA
   • 50 μL sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA. Adjust your DNA and water amounts to add 70 μL of volume total)
8. Vortex to resuspend cells.
9. Incubate for 30 minutes at 30°C.
10. Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min. The time may need to optimized for your strain and transformation conditions.
11. Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette.
12. Resuspend your cells in 200ul of water.
13. Plate 180ul of this culture onto a YPD plate and 20ul + 180μL H₂O onto a second YPD plate. Put at 30°C overnight.

Day 3

1. Replica plate from your YPD plates onto 5-FOA plates.

Colonies should be visible in 2-4 days. They may be smaller than you are used to. Restreak onto YPD for single colonies and check by colony PCR.

Notes

• This protocol is identical to the "normal" yeast LiAc transformation with TWO important changes (that we have found to be very important through trial-and-error):

1. The cells must be grown overnight and to mid-log for transformation in selective media so that you will not get spontaneous ura- mutations. If you don't grow your cells in ura- media we have found that almost all colonies that look like "transformants" on 5-FOA turn out to be spontaneous mutations in URA3.
2. The cells should be recovered on solid media (ie, on a YPD plate) and then replica plated the next day onto 5-FOA. Again, this prevents getting a lot of false transformants that just turn out to have mutations in URA3.

• You can alter the media recipe above to conform to how we make dropout media using YNB1 (1.1x) from the media facility:

1. Simply add the 100X 5-FOA and 25 mg uracil to .69g of KS-URA and 50mL of 20% dextrose (assuming 500mL total media volume.) Allow for ample time to dissolve.
2. Filter sterilize the above solution into the melted YNB1 only after it has cooled to about 55°C (warm, but not hot, to the touch.)
3. 25mg of uracil is equivalent to 1.25 mL of 100x uracil stock solution (2g/L)