Lab 5 Quantitative PCR and Epigenetics (continued)

Objectives:
  • Perform qPCR to measure gene expression
  • Develop dot blot (from last week) to measure DNA methylation




See lab 4 for procedure background
WesternBreeze® CHROMOGENIC IMMUNODETECTION (continued from last week)

General Guidelines

  • Avoid touching the working surface of the membrane, even with gloves. Use forceps
  • Work quickly when changing solutions as membranes dry quickly.
  • Add solutions to the trays slowly, at the membrane edge, to avoid bubbles forming under the membrane. Decant from the same corner of the dish to ensure complete removal of previous solutions.

1. Prepare 20 mL of Blocking Solution (prepared during lab last week)
  • a. Ultra filtered Water 14 ml
  • b. Blocker/Diluent (Part A) 4 ml
  • c. Blocker/Diluent (Part B) 2 ml
  • d. Total Volume 20 ml
2. Place the membrane in 10 ml of Blocking Solution in a covered, plastic dish.
3. Incubate for 30 minutes on a rotary shaker set at 1 revolution/sec.
4. Decant the Blocking Solution.
5. Rinse the membrane with 20 ml of water for 5 minutes, then decant. Repeat.
6. Prepare 10 mL of Primary Antibody Solution (1:5000 dilution)
  • a. Blocking Solution 10 ml
  • b. 5-MeC antibody 2 µl
  • c. Total Volume 10 ml
7. Incubate the membrane with 10 ml of Primary Antibody Solution for 1 hour. DURING INCUBATION SET UP qPCR
8. Decant primary antibody and wash the membrane for 5 mins with 20 ml of TBS-T. Decant and repeat three more times
9. Incubate membrane in 10 ml of secondary antibody solution for 30 mins. Decant
10. Wash the membrane for 5 mins with 20 ml of TBS-T. Decant and repeat three more times
11. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
12. Incubate the membrane in 5 ml of Chromogenic Substrate until color begins to develop (1-60 mins)
13. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
14. Dry membrane of clean piece of filter paper





Quantitative PCR

Supplies and Equipment:
  • PCR Plates (white); optically clear caps
  • 1.5 ml microfuge tubes (RNAse free)
  • Nuclease Free water
  • filter tips
  • Opticonthermal cycler
  • kim wipes
  • 2x Immomix Master Mix
  • SYTO-13 Dye
  • microfuge tube racks
  • ice buckets
  • timers
  • cDNA samples (student provided)


Procedure Background



see Lab 3 for PCR background



  • Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNAmolecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

  • The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

  • Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues.


Source: Wikipedia


qPCR PROCEDURE

Primer reconstitution (to be explained in lab)
The primers you designed are in a dehydrated state. After re-hydrating them, we will be making 100uM stock and a 10uM working stock to use for qPCR.

You will run each template (cDNA and RNA) in duplicate in addition to two negative controls (no template) for a total of SIX reactions

1. Prepare master mix: Prepare enough master mix for SEVEN reactions to ensure sufficient volume recovery.

For a 50μl reaction volume:
Component
Volume
Final Conc.
Master Mix, 2X (Immomix)
25µL
1x
Syto-13 dye (50uM)
2µL
2µM
upstream primer, 10μM
2.5μl
2.5μM
downstream primer, 10μM
2.5μl
2.5μM
Ultra Pure Water
16uL
NA

2. Add mastermix to wells of a white PCR plate
3. Thaw cDNA samples.
4. Add 2uL cDNA template to each reaction.
5. Add 2uL of ultra pure water to the negative control wells.
6. Cap the wells securely.
7. If necessary, spin the strips to collect volume in the bottom of the wells.
8. Ensure the lids are clean and place strips on ice.
9. Load the plate, verify the PCR conditions and start the run (this will be done by a lab research scientist).