Integrating Troublesome Tags in Yeast
(Bruce Goode 1-24-02)
This prot°Col is designed for efficient integration of Longtine-based PCR tags into yeast.
The general strategy is to use a TON of PCR product for a single transformation and you
should get 10-50 colonies.
DAY 1
1) Initiate 3-5 x 100µl PCR reactions. Use Taq DNA polymerase, the appropriate
Longtine plasmid, your 2 oligos, and complete 20 cycles.
2) Transfer ALL of the PCR products into a single 1.5ml tube. Remove 10µl into a
separate tube for gel analysis later.
3) Add 0.1 volumes 3M NaOAc and mix briefly by flicking tube. Then, add 2 volumes
100% ethanol and mix by vortexing.
4) Incubate on ice for 20-30 min.
5) Centrifuge (full-speed) in the microfuge, in the cold room, 20 min.
6) Important: BE THERE when the centrifuge run comes down. Carefully remove the
supernatant and discard. You should see a pellet.
7) Add (to the pellet) 1ml 70% ethanol, then centrifuge as above, 5 min.
8) Decant supernatant and invert tube over paper towels to air dry the pellet. This usually
takes about 20-30 min. If you are in more of a rush, instead speedvac the tube for 2-3
min. (no heat).
9) Resuspend the DRY pellet in 30µl TE, working up and down the sides of the tube to
get all of the DNA solubilized. Remove 2µl DNA for analysis on a gel (compare to
the 10µl you saved in step #2 above).
10) If you have at least 1µg DNA, pr°Ceed.
DAY2
11) Grow 50ml of yeast in YPD to an OD600 = 0.5-1.0.
12) Pellet cells in clinical centrifuge 3 min., 2500 rpm. Decant media. Resuspend cell
pellet in 1ml ddH2O using a P1000 and transfer to a 1.5ml tube. Pellet cells in
microfuge, 5 sec. full-speed. Remove supernatant.
13) Resuspend the cell pellet in 300µl of TE/0.1 M LiOAc.
14)
It is important that the DNA is freshly boiled 2-5 min., then chilled on ice for 5
min. immediately before use. For calf thymus DNA, do NOT boil. Just thaw and
use.
15) Add 15µl ethanol precipitated PCR product, and flick to mix.
16) Transfer 170µl of this mix to each of 3 tubes (1.5ml). To each tube, add 1ml TE/0.1
M LiOAc/40% PEG-3350 [for 3ml, mix 300µl 1M LiOAc, 300µl 10X TE, and 2.4ml
50% PEG-3350 by vortexing inverted in a 15ml tube].
17) Incubate 60 min. at 30°C, spinning (place the 1.5ml tubes in culture tubes).
18) Heat shock in a 42°C water bath, 10 min.
19) Pellet cells in microfuge, 10 sec., full speed. Aspirate supernatant.
20)
2O.
total of 3 plates).
Plate immediately (one tube per plate –