ELISA test plate protocol

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					Biotechnology Methods and Techniques                                 Revised LLP, 2010

ELISA Lab: Test plate

Step 1: Obtain a 96-well PRACTICE microtiter plate.

Label the plate with your name and date on the edge of the plate.
Put a line with marker between columns 6 and 7.

Step 2: Using an 8-channel pipettor, add 180ul of YELLOW TEST SOLUTION to every
well of your plate.

Step 3: One at a time, add 20ul of NEUTRAL RED to each of the wells in column 1 and
each of the wells in column 7.

Mix each sample by pipetting up and down gently 3-4 times, but don’t blow bubbles into
the sample.

Step 4: Put a piece of tape over column 7 of the plate. Using the 8-channel pipettor with
8 NON-STERILE tips in place, use the method below to do a 1:10 serial dilution of
samples in column 1 to column 2, column 2 to column 3, 3 to 4, 4 to 5, and 5 to 6.
Remove 20 ul of solution from column 6 and throw it away. STOP! Get new tips.

Take off the tape. Repeat those steps, starting with column 7, diluting to 8, 9, 10, 11 and
ending with column 12. Remove 20 ul of solution from column 12 and throw it away.

DILUTION METHOD: To do the 1:10 serial dilutions (20 ul : 200 ul is the same as
1:10), set the multichannel pipettor to 20 ul, take 20 ul from all of the filled wells in
column 1 and add to the wells in column 2; mix by drawing up and down gently (no
bubbles) 3-4 times. Using the same tips, continue by moving 20 ul from column 2 to
column 3, etc through column 6. Use one finger to mark the column you are working
with so you don’t lose track. Throw away the 20 uL from column 6 – DO NOT MOVE

CHECK: If you’ve done this right, columns 1 and 7 should be bright red and columns 2
and 8 should be light orange. All the rest should be yellow. All columns should have
identical amounts of liquid (180 uL) – you can check by looking at your plate from the

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