1.  Not1/Snf1 double digest of p416-BCCP secretion vector. (Enough for > 200 transformations. It is very important to insure complete digestion with both enzymes)

A. 5ul NEB#2, 10ul 1ug/ul vector, 10 units Not1, nuclease-free H2O to 50ul. Incubate 2hrs, 37oC.

B.  5 µl NEB#2, 10ul 1ug/ul vector, 10 units Sfi1, nuclease free H2O to 50ul. Incubate 2hrs, 50oC.

C.  Run 0.5 µl each digest on agarose gel to assure yourself that both enzymes are cutting. Run some uncut vector for comparison. Continue digests while gel is running.

D.  If both enzymes are active and digests are complete, or almost complete, add 10 units of Sfi1 to the Not1 digest and incubate 3-4 hrs at 50oC. Similarly, add 10 units of Not1 to the Sfi1 digest and incubate at 37oC.

E.  Mix both digests, add 10 units more of each enzyme and continue the digest overnight at 37oC.

F.  Extract with phenol/chloroform/isoamyl alchohol (25:24:1).

G.  Add 2 volumes of 100% EtOH, put at –80oC for 15-30 min, centrifuge 10 min 13000 rpm. Air dry pellet. Do not overdry.

H.  Resuspend in 100 µl -200 µl Qiagen EB. It may take 16-20 hrs to resuspend completely.

I.  Remove and aliquot, dilute 1:100 and measure OD260 and 280.

2.  Colony PCR of scFv from pAGA2.

A.  Patch the scFv on a SD+CAA plate. Grow 1-2 days at 30oC. Use patches within 3 days.

B.  B. Scrape up a small amount of cells, about the size of a pinhead on a clean micropipet tip. Resuspend in 20ul 20mM NaOH in a PCR plate. I use a plate so that the tubes can stand upright in the microwave without a rack which might shield the lysate.

C.  Microwave 3 min full. Cool to room temperature. Add 3 µl lysate to 47ul PCR mix described below.

D.  PCR mix:

25.5µl of nuclease-free water
1 µl 10mM dNTP mix
1 µl 10uM Forward Ggt tct ggt ggt gga ggt tct ggt ggt ggt gga tct g
1 µl 10uM Rev gag acc gag gag agg gtt agg gat agg ctt acc gtc gac caa gtc ttc ttc aga aat aag ctt
5 µl Colony PCR buffer (recipe below)
5 µl 1mg/ml BSA
8 µl 50mM MgSO4
0.5 µl Platinum Taq HiFi polymerase

E.  Colony PCR mix: 0.1M Tris-HCL, 0.5M KCl , pH 8.3, filter sterilized.

F.  PCR protocol:

1. 94oC 3min
2. 94oC 1min
3. 55oC 1.5min
4. 72oC 3min
5. Go To 2 35X
6. 72oC 5min
4. 4oC forever
END


3. Gel purification of PCR fragment (necessary since there are often several PCR products)

A. Run PCR mix on ) 0.8% agarose gel.

B. Cut out band. Clean with QiaEx kit.

4. Colony PCR from pNL9 or pTOR001 to confirm insertion.

A. Patch to SD+CAA+TRP. Grow 1-2days

B. Everything is the same as 2. above expect that the PCR mix is different:

32ul nuclease-free H2O
1ul 10 mM dNTP mix
1ul 10 µM Gal1 (primer1)
1ul 10 µM Cyc1 (primer2)
5 µl Colony PCR buffer
5 µl 1mg/ml BSA
1.5 µl 50mM MgSO4
0.5 µl Taq DNA

5. Transformation with Not1/Sfi1-cut vector and PCR fragment. (Note: The outgrowth after heat-shock is essential for recovering transformants with inserts. Other important factors: The cell density at the start of the transformation, the pH, denaturing the carrier DNA, the polymerization number of the PEG and adding DMSO. )

A. Make a stock of YVH10 in 2xYEPD: Inoculate from the freezer culture into 5ml YEPD and grow overnight on the roller at 30oC. Store the stock at 4oC. It can be used for 2 months or more, although viability will decrease with time.

B. Grow cells in 2xYEPD at 30oC overnight to stationary. Late log phase. Use the fresh ON culture to inoculate a flask of 2xYEPD at a 20x dilution. Shake at 30ºC for 3-5hrs.

C. The cell density should be 5x106 to 2x107 cells/ml. Transformation efficiency falls off sharply beyond those limits though you will probably some transformants even if the density is not ideal. You can’t get away with using a smaller volume of a thicker culture. If the cells have overgrown, you should dilute back to about 1-2x106 and let the cells go through several doublings (about 5hrs)

D. Transfer the cultures to centrifuge tubes and centrifuge 5min 3000 rpm.

E. Decant. Resuspend the cells in 10ml sterile water.

F. Centrifuge as before. Resuspend in 1XLiAc/TE, 1ml for each transformation that will be done. (Remember the no insert control.) . Let sit at RT 10 minutes. (note: pH is very important. All solutions should be at 7.5.)

H. Transfer 1ml of LiAc treated cells to microfuge tubes. Centrifuge 1 minute 6K rpm. Remove SN thoroughly with pipetman. Put the pellets on ice.

I. Layer cold 0.5ml 40%PEG 3350, 100mM LiAc, 1mM EDTA, pH7.5 over the pellet. DO NOT resuspend.

J. Layer at the top of the PEG: 20ul per transformation of denatured salmon sperm DNA, 10mg/ml, 0.2 ul Not1/Sfi1-cut vector, approx. 100ug/ml. and 10ul gel purified insert . Freshly denatured carrier DNA for best results. DO NOT add DMSO.

L. Vortex 15 seconds, top speed to resuspend the pellet. NOTE: It is OK to vortex but don’t overdo it.

N. Heat shock at 420C for 30 minutes (Note: 30-45 min heat shock give about the same transformation efficiency, 15 and 60 minutes give less.).

O. Centrifuge 5min 6000rpm in microfuge. Don’t overspin. The cells will form clumps that are practically impossible to get rid of.

P. Resuspend pellet in 500ul 2xYEPD by vortexing.

Q. Put microfuges tubes into 10ml tubes and install them on the roller 3-16hrs at 300C (or you can let them sit if you invert them from time to time to keep the cells from settling out.)

R. Centrifuge 5min 6000rpm. Resuspend in sterile 200ul H2O. Do a 1:10 and 1:100 dilution if you are going to pick individual colonies. Spread 0.1 ml of undiluted and 1:10 and 1:100 dilutions on 2 SD+CAA+TRP or C-ura plates. Incubate at 300C. Transformants will be visible in 2 days, ready to pick in 3-4 days.

Solutions for transformation

Stocks—may be stored at room temperature indefinitely

1M LiAc pH7.5: 102 gm/l adjusted to pH7.5, filter sterilized

10X TE 7.5: 0.1mM (12.1gm/l) + 0.01M EDTA (3.7 g/l) pH to 7.5 w/HCl, filter sterilized

1XLiAc/TE: 1:10 dilutions of above

40% PEG/0.1M LiAc/1X TE pH7.5: 400g/l, 100ml 1M LiAc, 100ml 10X TE pH7.5, filter-

sterilized.

Media

Liquid YEPD

SD+CAA+TRP plates or C-ura plates

2