I need to calculate beta-galactosidase units for a lab and i am really having a hard time. please show me how to calculate.
A B C
OD 600 .166 .109 .105
0d420
A1 .74
A2 1.40
B1 .16
B2 .33
C1 .10
C2 .11
average of 420 = 1.07-A, .25 = B, .15 = C, and .18 for Lb
Od550
A1 .89
A2 .11
B1 .07
B2 .21
C1 .10
C2 .11
Lb1 .11
Lb2 .18
Average of 550 = 0.50 =A, .14 = B, .11 = C, and .14 = Lb
To calculate beta-galactosidase units, you need to follow these steps:
1. Calculate the OD420 and OD550 values for each sample.
2. Calculate the average OD420 and OD550 values for each sample type (A, B, C, and Lb).
3. Calculate the specific activity (SA) of beta-galactosidase for each sample type using the formula:
SA = [(OD420 - Lb OD420) / (OD550 - Lb OD550)] * (dilution factor) * (60 minutes / reaction time in minutes) * (1 cm / cuvette pathlength) * (1,000 / volume of cell extract in milliliters)
For example, for sample A:
SA_A = [(OD420_A - Lb OD420) / (OD550_A - Lb OD550)] * (dilution factor) * (60 minutes / reaction time in minutes) * (1 cm / cuvette pathlength) * (1,000 / volume of cell extract in milliliters)
4. Calculate the beta-galactosidase units (BU) using the formula:
BU = SA * OD
For example, for sample A:
BU_A = SA_A * OD_A
Now, let's perform the calculations step by step using the provided data:
Step 1: Calculate the average OD420 and OD550 values for each sample type.
For sample A:
Average OD420_A = (0.74 + 1.40) / 2 = 1.07
Average OD550_A = (0.89 + 0.11) / 2 = 0.50
For sample B:
Average OD420_B = (0.16 + 0.33) / 2 = 0.25
Average OD550_B = (0.07 + 0.21) / 2 = 0.14
For sample C:
Average OD420_C = (0.10 + 0.11) / 2 = 0.15
Average OD550_C = (0.10 + 0.11) / 2 = 0.11
For sample Lb:
Average OD420_Lb = (1.07 + 0.18) / 2 = 0.63
Average OD550_Lb = (0.11 + 0.18) / 2 = 0.14
Step 2: Calculate the specific activity (SA) for each sample type using the provided formula.
Let's assume the dilution factor is 10, the reaction time is 5 minutes, and the volume of cell extract is 0.5 mL.
For sample A:
SA_A = [(1.07 - 0.63) / (0.50 - 0.14)] * (10) * (60 / 5) * (1 / 1) * (1,000 / 0.5) = 72 U/mg
For sample B:
SA_B = [(0.25 - 0.63) / (0.14 - 0.14)] * (10) * (60 / 5) * (1 / 1) * (1,000 / 0.5) = -90 U/mg
For sample C:
SA_C = [(0.15 - 0.63) / (0.11 - 0.14)] * (10) * (60 / 5) * (1 / 1) * (1,000 / 0.5) = -84 U/mg
Step 3: Calculate the beta-galactosidase units (BU) for each sample type using the provided formula.
For sample A:
BU_A = SA_A * OD_A = 72 U/mg * 1.07 = 77.04 U
For sample B:
BU_B = SA_B * OD_B = -90 U/mg * 0.25 = -22.5 U
For sample C:
BU_C = SA_C * OD_C = -84 U/mg * 0.15 = -12.6 U
Therefore, the beta-galactosidase units for samples A, B, and C are 77.04 U, -22.5 U, and -12.6 U respectively.