Difference between revisions of "Yeast Transformation (Clontech kit)"

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Materials
Materials


3B Bovine Alpha S2  - 53.4 nano-gram/micro liter
3B Bovine Alpha S2  - 53.48 nano-gram/micro liter
6b Bovine Beta A2  - 90.01 nano-gram/micro liter
6b Bovine Beta A2  - 90.01 nano-gram/micro liter
7  Bovine Kappa    - 31.87 nano-gram/micro liter
7  Bovine Kappa    - 31.87 nano-gram/micro liter

Revision as of 16:15, 8 December 2014

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 - 53.48 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.

3. Make sure that the trnadofrming DNA (3b, 6B, and 7) are sufficient concoentrated because we need 150-200ng DNA concentration (1.0 - 1.5 micro liter per reaction)

We transformed 3 genes (3B, 6B, and 7)

Tube 1 Tube 2 Tube 3 Tube 4
3B 6B 7 Control
Denatured Carrier DNA 5 ul 5 ul 5 ul (we added 3ul)
Transformed DNA 4 ul 2 ul no
Yeast transformation mix 91 ul 93 ul 95ul
Total 100ul 100 ul 100 ul


3A Bovine Alpha S2 = 53.48 ng/ml we need 150-200ng per ml so took 4ml So 53.48 * 4 = 213.92

6B Bovine Beta A2 = 90.01 ng/ml, took 2ml So 90.01 * 2 = 180.02

7 Bovine Kappa = 31.87 ng/ml took 6ml So 31.87 * 6 = 191.22

Results: no colonies on every plate including - control