Serial Dilution

Step 1

Make table

Step 2

get 6 cuvettes and label them 0-4 and BL for blank

Step 3

Add how many ever mL of bromophenol blue (8mg/ml) stock solution to the cuvette labeled 0 using p-1000 (twice)

2mL

2000

step 4

fill each cuvette labeled 1-4 with 1mL (1000) of 1X TAE BUFFER using P-1000

step 5

switch vortexer to on and set the dial to 6

step 6

transfer 1mL (1000) of stock solution (transfer volume) from cuvette 0 to 1 (vortex to mix)

step 7

uncap cuvette 1 and transfer 1mL (1000) from cuvette 1 to cuvette 2. cap and mix

step 8

repeat the transferring and mixing procedure through cuvette 4

step 9

take 1ml (1000) from cuvette 4 and discard into rinsate beaker

Step 10

PREPARE YOUR BLANK

step 11

to prepare blank: add 1ml (1000) of 1X TAE buffer to the cuvette labeled BL (use TAE buffer as blank)

step 12

cap all cuvettes and set aside until ready to read absorbance

Step 13

prepare UNKNOWn sample A of bromophenol Blue for Absorbance measurement

step 14

Label 1 cuvette A

Step 15

take the tube labeled Unknown A and transfer (2mL) p-1000 twice to the labeled cuvette A

step 16

cap and ready to measure

step 17

grab lab quest

the absorbance of a sample is

directly proportional to the concentration of material in the sample

step 18

connect labquest to spectrovis using USB plug

step 19

power on labquest wait 1-2 minutes

step 20

go to meter screen by pressing clock type button in the upper left corner of the screen

step 21

go to sensors in the menubar and press CALIBRATE from drop down menu wait 90 seconds for warm up to finish.

step 22

take BL and wipe the cuvette for dust then place it in the cuvette holder of the spectrovis and press FINISH CALIBRATION. when complete press okay

step 23

Go to sensors and select data collection. CHANGE MODE from FULL SPECTRUM TO TIME BASED and press OK

step 24

tap the red box on the screen and select CHANGE WAVELENGTH from drop down

step 25

enter value 550 and press okay. NOW YOU HAVE THE ABSORBANCE of your solution

step 26

remove the BL and take cuvette 0 and place in spectrovis

step 27

to see absorbance begin data collection by pressing green arrow for 10 seconds then hit stop

step 28

go to Analyze in the menu bar and select Statistics from drop down menu. Select your run. Record the average (MEAN)

step 29

remove cuvette 0 and put cuvette 1 press play

step 30

collect the absorbance for each cuvette and record them

step 31

record the absorbance of UNKNOWN A

step 32

use the absorbance number from UNKNOWN A to calculate the concentration

concentration formula

Cu= Au*CS/ As

Cu

concentration of the unknown

Au

absorbance of the unknown

Cs

concentration of the standard

As

absorbance of the standard

concentration units

mg/ml