BIOL 111 Lab 2 Measurement and Standard Curve Lab Activity 1 Using Standard Curves to Calculate Unknown Concentrations Both Part A and Part B should be set up and performed simultaneously Ideally perform the serial dilution and initial testing of the milk first then set up the standard curve and measure the appropriate dilution of milk Y e o y a e s e y k s n t r i r u s d y d m r s d c r m u h e m u w l n Page 1 of 7 o h o u l e a s u r o u i l a m p l h p e c t r o p h o t o m e t e m m e d i a t e l f t e y o a v e a s u r e o u t a n d a r u r v e Y o i l e e d e d C e S e 1 calculating the volume of BSA Bradford Reagent and t A T Complete T 0 15M NaCl necessary to make a 1 0 mL solution with the final concentration shown in each row You will be required to show your TA your completed table at the start of lab u s P d h n a h t g i e T d t e t n o o w e 1 a e s n t m u y f d i n B E L t t e s t o r a f l b s o t t i t h s 2 Label your tubes cuvettes at the top of the tube with a sharpie Make sure not to label towards the bottom or in the way of the light source of the spectrophotometer See image to the right 3 To each cuvette add Bradford Reagent first then NaCl and finally BSA Put the tip of the pipette tip into the reaction when delivering small volumes to your cuvette Remember to remove the tip from the reaction before releasing the plunger after delivery 5 Incubate the tubes for approximately 3 5 minutes at room temperature 4 Mix well by carefully and gently flicking the cuvette 6 While incubating set the wavelength lambda of the spectrophotometer to 595 nm This may have already been done but you should check that the instrument is set correctly see figure below y w 7 G 8 Blank zero the spec with What components should be in your blank cuvette to be used for h a K e w e e h c r o e t f e measuring your BSA solutions in the spectrophotometer Page 2 of 7 P a r h t a n d a r u r v a b l Y o h o u l a v a b l l r e a d i l l e E F O R A B 1 l u n u r h p e c t r o p h o t o m e t e h t a r a h a a e n o u g i m a r p e n t l i p h x t e r i o a c u v e t t i t i m w i p e 9 Measure the absorbance of each sample Place the cuvette in the spectrophotometer in the slot for cuvettes and gently close the lid DO NOT FORCE THE LID CLOSED Wait for the numbers to stabilize and record the value then move on to the next tube 10 Fill in T t B D e 2 with the absorption measurements of your known BSA standard solutions s a d D g t e C n o f t e U n 1 Obtain a sample of milk from the TA and record the identity of the milk 2 Make 10 fold serial dilutions of your milk sample to arrive at a final dilution of 1 1000 To do so a Add 20 L milk to 180 L DH2O to create a 1 10 n 2 dilution in a microcentrifuge tube L m k i 0 L l v e i s 1 b Mix well by capping the tube and flicking it vigorously s i n t e n t s p i f y u d t m x w l t h 110 110 1100 c Make a second 1 10 dilution using 20 L from the dilution that you just made 180 L H2O as shown in the figure below for 1 100 dilution d Repeat dilutions until you get 1 1000 Page 3 of 7 a b l P a r i l u t i o n n e t e r m i n i n h o n c e n t r a t i o h n k n o w 2 0 i l 0 f i n a o l u m 1 0 W h a a p p e n h e x t e o o n i e l h e r e Use T r c 3 Use 10 L of each dilution in a Bradford assay and observe the color change Did the cuvette immediately turn bright blue might be outside the linear range and would not be a good candidate If the color matches one of your middle standard curve samples you are within range Which dilution matched the middle of your standard curve sample 4 Use this dilution of milk to proceed and measure it using 5 10 20 and 40 L of the diluted milk e 3 to record your data and use the blank from Part A B e s e t o w e t e e r o f e w h a K O e y u a e f d g g y r a e r p e t n o f a d u g t e Drawing a Standard Curve Now that we have collected the absorbance values of each of the different known BSA solutions standards in Part A of Lab Activity 1 we must plot the standard curve Generate a scatter plot with concentration on the X axis and absorbance on the Y axis to turn in A line of best fit must then be put on the data set Note that this is not the same as a line graph as our line will not connect each of the data points but should best represent the trend at which the data is moving By using the line of best fit we can determine the concentration of our milk solutions An important question we must first answer is whether we believe that our standard curve is accurate Errors in measurement could lead to an incorrect measurement of our milk sample We can statistically test the quality of our standard curve by calculating the regression coefficient R2 This calculation will tell us how close our …
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