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 - 53.48 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 (should be microliter) 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 (should be microliter) 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 (should be microliter) 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 reannealing.
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 concentrated 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