Difference between revisions of "Yeast Transformation of All Bovine Casein Inserts"
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We used the Quick & Easy Yeast Transformation Mix Protocol-at-a-Glance | 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 | 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. | 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 | C. Denature the yeast maker (salmon sperm) carrier DNA at 20ul | ||
D. Incubate at 45 degrees C for 65-70 min in a PCR | 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) | |||
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 | F. Plate 100ul of each dilution onto selective media CMURA- plate | ||
To make transformation mix to add using | To make transformation mix to add using | ||
1. 5ul denatured yeast maker carrier DNA | 1. 5ul denatured yeast maker carrier DNA | ||
2. 150-200ng of target DNA | 2. 150-200ng of target DNA | ||
3. Fill to 100ul with quick n easy yeast transformation mix | 3. Fill to 100ul with quick n easy yeast transformation mix | ||
We transformed 5 genes (3B, 6B, 7, 8 and 1) | 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. | 250 ml of CM minus URACIL liquid media was prepared in a glass bottle using 8g of powder and 4g Agar. | ||
Latest revision as of 05:34, 13 January 2015
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: