Yeast Transformation (Clontech kit)

Tuesday November 18 2014 Yeast transformation (Clontech kit) with rest bovine gene (minipreped from transformed bacteria) Participants: Nikola, Johan, and Meenakshi

Check cone of minipreped plasmid

Materials

3B Bovine Alpha S2 - 53.4 nano-gram/micro liter 6b Bovine Beta A2 - 90.01 nano-gram/micro liter 7 Bovine Kappa - 31.87 nano-gram/micro liter

A. Take 1 colony from plate and resuspend in 400 milliliter of distilled water

B. Voretex the tube to make homogenized sample and centrifuged at 3000g at 3 minutes and pool out the supernatant carefully.

C. Add 100 milliliter of transformation mix from step 3 and vortex 3 x 1 seconds to resuspend the cells into mix

D. Incubate at 45 degrees C for 65-70 min.

E. Dilute the incubated sample ten-fold and 100 fold (that is 100 incubated sample + 900 micro liter water and again dilute it in 9 ml distilled water total volume would be 10ml)

F. Take 100 milliliter the diluted sample and plate it on URA - plate and wrap the plate in foil as it is light sensitive and put it 30 degrees C oven

To make transformation mix to add

1. Denature the yeast maker (salmon sperm) carrier DNA at 95 degrees C for 5 minutes. Place tube on ice to prevent reanneling.

2. Thoroughly vortex the quick easy yeast transformation mix tube before use, as precipitate may form. If frozen, allow it to thaw and then vortex.