Yeast Transformation of All Bovine Casein Inserts

10 January 2015: Yeast Transformation of All 5 Bovine Casein Inserts

Location: BioCurious

Participants: Lafia, Johan, Meenakshi, Maria, Gabby, Nikola, Joseph, Emi, Aaron, Julianne, Gabby

Notes: Emi

Aims: To see the casein gene expression and the confirmation is the SDS Page

Materials: 7 CMURA- plates

Pipette tips

Centrifuge

Incubator

PCR

7.5 grams of Agar

Bov alpha S1 (1)

Bov Beta B (8)

Bov Alpa S2 (3B)

Bov Beta A2 (6B)

Bov Kappa 7

Methods:

We used the Quick & Easy Yeast Transformation Mix Protocol-at-a-Glance

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

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

C. Denature the yeast maker (salmon sperm) carrier DNA at 20ul

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

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. Plate 100ul of each dilution onto selective media CMURA- plate

To make transformation mix to add using

1. 5ul denatured yeast maker carrier DNA

2. 150-200ng of target DNA

3. Fill to 100ul with quick n easy yeast transformation mix

We transformed 5 genes (3B, 6B, 7, 8 and 1)

250 ml of CM minus URACIL liquid media was prepared in a glass bottle using 8g of powder and 4g Agar.

'	3B	6B	1B	7A	8B	Control
	Bovine alpha s2	B. Beta A2	B Alpha S1	B Kappa	B Beta 8
Denatured Carrier DNA	5ul	5ul	5ul	5ul	5ul	3ul
Transformed DNA 3B, 6B, 7, 1B, 8B	4ul	1.5ul	1.5ul	3ul	1.7ul	-
Yeast transformation mix	91ul	93.5ul	93.5ul	92ul	93.3ul	97ul
Total = 100ul

Plates streaking: