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  • GO BACK AND USE THE "NEWER" PROTOCOL*


NOTE: BEFORE YOU START I SUGGEST YOU MAKE THE REAGENTS AT THE END BEFORE YOU START

For each transformation...

1) Pellet yeast from 3 ml of a mid-log culture at 3000 rpm for 2 min. (see below for ideas on how to grow yeast).

2) Aspirate supernatant. Resuspend in 3 ml 0.1 M LiOAc and re-spin at 3000 rpm for 2 min.

3) Aspirate Supernatant. Resuspend in 1 ml 0.1 M LiOAc, tansfer to a microfuge tube, and re-spin in a microfuge tube at 3000 rpm for 2 min.

4) Aspirate using P1000. Resuspend in 30 ul 0.1 M LiOAc + carrier DNA1. (In the microfuge tube).

5) Add up to 15 ul DNA (e.g., PCR product that had been purified using a Qiaquick column).

6) Sit 15 min. at room temp.

7) Add 150 ul Goop2.

8) Sit 30 min at RT.

9) Incubate in a 42 water bath for 15 min.

10) Spin yeast out slowly (2000 rpm, 2 min).

11) Aspirate supernatant and resuspend yeast in 0.5 ml YPD.

12) Leave at 30C (incubator, not shaker- just leave the tubes in a rack) for 1 hr (or 4 hr if the phenotypic lag of a drug-resistance marker needs to be overcome). For a particularly low background growth on a dropout selection plate, can gently re-spin and resuspend cells in water.

13) Gently re-spin at 2000 rpm for 2 min and resuspend in 500 ul of water.

14) Plate. We should have about 500ul- plate to three selective plates: 85ul, 165ul, 250ul (In other words, 85ul, 165ul, and whatever is left).

When only one a couple of transformations per parent strain are being done, can transform in the morning since dilutions of an overnight YPD culture can be done when the overnight is started (e.g., 4 ml of innoculated YPD can be diluted 5x, 25x, 125x and 625x). In the morning, the culture that is closest to an OD of 0.5 to 2 is used and rest discarded.

When doing more than a couple of transformations per parent strain, 25 ml or more of YPD in a standard 125 ml Wheaton bottle is innoculated with 2 ml late log overnight culture (make several dilutions of the 2 ml culture when it is started and discard the ones you don’t need in the morning).

1 For 10 transformations add 0.3 ml 0.1 M LiOAc to 30 l (freshly boiled and iced) 10 mg/ml salmon sperm DNA.

2 For 10 transformations freshly mix 0.4 ml 0.5 M LiOAC and 1.6 ml Boone PEG (Boone PEG: dissolve 150 gm PEG3550 in 165 ml ddwater and filter sterilize)



protocol originally from getyourscienceon wiki

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