Jacobs:Protocol Real-Time PCR

Overview

Protocol for Real-Time PCR

Materials

• GeneAmp RNA PCR Core Kit (Part# N808-0143, Applied Biosystems)
• PCR reaction tube
• Centrifuge
• Liquid Wax
• Thermal cycler
• RNase free Microcentrifuge tubes
• RNase free H2O
• Taqman PCR Master Mix (Applied Biosystems 4304437)
• 20X Primers and Probes (Applied Biosystems)
• 384-well plate (Applied Biosystems 4309849)
• Optical cover (Applied Biosystems 4311971)
• Light mineral oil (Fisher M5904)
• Real Time PCR machine
• Various pipet tips and pipetter
• RNase away
• Marker

Procedure

1. Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
   1. Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
   2. In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
   3. Pipette 60 µl of BSA from tube #1 into #2. Pipette up and down a dozen times or until you think it is properly mixed. Then take 60 µl from tube #2 and put it in tube #3 and pipette up and down. Continue this process through tube #5 (this will leave tube #5 with 120 µl)
2. Prepare BCA Working Reagent
   1. For the total volume of working reagent calculate:
      • (# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl))
   2. To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
3. Prepare your samples
   1. Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)
      1. Put 60 µl of your sample in tube S1
      2. Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
      3. Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
4. Prepare your Microplate
   1. Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
   2. Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
   3. Incubate plate at 37C for 30 minutes
   4. Remove plate and measure the absorbance at 562 nm on a plate reader
   5. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
   6. Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl. Use the standard curve to determine the protein concentration of each unknown sample

Notes

Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops

Contact

• Originally prepared by CRJ-EJC 1/3/06

or instead, discuss this protocol.