Lab 3: Reverse transcription and qPCR, analyze qPCR (via computer data and agarose gel) Primer design, and Protein Extractions

Please print these lab materials before each lab.

Lab Objectives

Supplies and Equipment:

Procedure background

A reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribed single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this. The resulting cDNA is more stable than RNA (which degrades quickly) can be used for downstream applications such as measuring gene expression. Partial nucleotide sequences of cDNAs are often obtained as expressed sequence tags. (This text has been adapted from Wikipedia)

REVERSE TRANSCRIPTION

  1. Mix your stock RNA sample by inverting tube several times.

  2. In a 0.5 ml PCR tube labeled with your initials and “cDNA” combine the following:

    1. 5 μl of YOUR total RNA (extracted and quantified in lab)
    2. Make a master mix with the following reagents (volumes listed are for a single reaction; calculate how much of each reagent you will need based on the number of reactions you'll be running)
    3. 0.5 μl of oligo dT (for eukaryotes) OR 0.5 μl of random primers (for prokaryotes)
    4. 12.25 μl of nuclease free H2O
    5. Add 12.75 μl of the master mix to each of your PCR tubes containing 5μl of your RNA.
      .

  3. Incubate the mixture for 5 min at 70°C in the thermalcycler then immediately transfer to ice. Briefly centrifuge your tube.

  4. Make a master mix with the following reagents (volumes listed are for a single reaction; calculate how much of each reagent you will need based on the number of reactions you'll be running)
    1. 5 μl of M-MLV 5X Reaction Buffer
    2. 1.25 ul of dNTPs
    3. 0.5 μl of M-MLV RT
    4. 0.5 μl of nuclease free H2O
  5. Add 7.25 μl of the master mix to each reaction, mix and briefly centrifuge reaction tubes.
  6. Incubate the mixture for 60 min at 42°C (for oligo dT) or at 37°C (for random primers) and then heat inactivate at 70°C for 3 min on the thermalcycler.
  7. Spin down the sample in a desk top centrifuge.
  8. Store on ice or at -20°C

Quantitative PCR

Supplies and Equipment:

Procedure Background

ROBERTS_Realtime PCR_SLIDES

qPCR PROCEDURE

You will run each template (cDNA) in duplicate in addition to two negative controls (no template) - calculate how many reactions this will be!

1. Prepare master mix: Prepare enough master mix for your number of reactions +1 to ensure sufficient volume recovery.
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2. Add mastermix to wells of a white PCR plate
3. Thaw cDNA samples.
4. Add 1uL cDNA template to each reaction.
5. Add 1uL 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. (I like to wipe the lids with a clean kimwipe)
9. Load the plate, verify the PCR conditions and start the run (this will be done by your TA).

PCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 15 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Making an agarose gel

Supplies and Equipment:

AGAROSE GEL POURING PROCEDURE

  1. Weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
  2. Microwave solution for ~ 3 minutes. Keep an eye on the solution so that it does not boil over. You want the solution to be clear - no precipitate and no bubbles.
  3. Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
  4. Mix thoroughly by swirling, then pour into gel tray.
  5. Add gel combs. Using a clean pipet tip, pop any bubbles that could get in the way of your PCR product.
  6. After gel is set, wrap in plastic wrap (label with your initials and date) and place gel in the fridge if not using immediately.

Agarose Gel Electrophoresis

Procedure Background

ELECTROPHORESIS PROCEDURE

  1. Place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
  2. Remove combs from wells
  3. Load 7uL 100bp ladder in far left lane
  4. Load 20uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
  5. Run gel at ~ 100V for ~ 1hr
  6. Visualize the gel on the UV transilluminator
Experiment Mock-up
  1. Now that you know what your biological question is and what controls and replicates you'll need for this experiment, you'll need to do a mock-up of the experimental containers so that the system will be ready later in the week when your experiment starts.
  2. A number of containers, heating elements, water baths, airstones and pumps are available. Work as a group to decide how the experiment will be organized.

Primer Design

Primers, or oligonucleotides (oligos), are short stretches of synthetic DNA that are used most commonly for PCR and DNA sequencing. They direct DNA polymerases to specific regions on larger DNA molecules for amplification. They are designed in pairs to amplify DNA in the forward and reverse directions. Oligos are custom synthesized by various manufacturer's to contain the precise sequence requested by the customer. For a good introduction to the theory of primer design procede to this link.

Photo source: <http://bioweb.uwlax.edu/genweb/molecular/seq_anal/primer_design /primer_design.htm>

Here is a breif list of things to take into consideration when designing primers. Although none of these are absolute, they will help ensure your primers will hybridize to your target sequence with the best efficiency.

1. Design your primers to be within 18-30 bases in length.
2. The melting temperature (Tm) of primers should be within 2C of each other.
3. Avoid primer dimers and primer hairpins
4. Avoid high G/C stretches, particularly at the 3' end
5. G/C clamp at 3' end of primers.

Primer design is most commonly done via computerized means and the algorithms used take the above rules into consideration. Of course, the user always has the opportunity to adjust the parameters that define how primers are designed by the software. There is a great deal of software available for primer design. Two commonly used primer design tools are NCBI Primer and Primer 3. The software will allow you to enter a full DNA sequence and then define what region(s) you would like to amplify, the ideal size of the amplicon (PCR product), the ideal length of the oligos, etc. However, often the preset conditions are already optmized to Additionally, after you have selected some proposed primers, you can compare melting temps, G/C content, primer dimer/hairpin probabilitites, etc.

We will go over the process of designing primers in more detail in lab.

Protein Extraction and Analysis Part 1

Supplies and Reagents

Procedure Background

This section provides an explanation of the methods being used and provieds some essential background information.

IMPORTANT SAFETY NOTES

PROTEIN EXTRACTION PROTOCOL

  1. Record the weight of your tissue that has been denoted on the tube.
  2. Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker.
  3. Add 500 ul of CellLytic MT solution to the 1.5mL snap cap tube containing your cut piece of frozen tissue.
  4. Homogenize the tissue with a sterile disposable pestle.
  5. Close the tube and invert the tube several times.
  6. Please find a few other people at or near this same stage and form a group for this step. Spin the tube in a refrigerated microfuge for 10 mins at max speed.
  7. While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date.
  8. Carefully transfer supernatant (the clearish liquid on top) to labeled tube and store tube on ice.

PROTEIN QUANTIFICATION PROTOCOL

  1. Lable a fresh 2 mL screw cap tube withe the word "Protein", BA (for Bradford Assay), your initials, and today's date.
  2. Dilute an aliquot of your protein sample 1:2 by pipetting 15uL of your protein sample into the 2 mL screw cap tube and the pipetting 15uL of DI water. Mix well by pipetting. Note: this dilution step is performed to ensure the sample absorbance falls within the range of the standard curve
  3. In a second 2 mL tube pipette 30uL of DI water (this tube will serve as your blank). Label tube as 'blank'
  4. To both tubes add 1.5mL of Bradford reagent. Tip: Pippet 1000ul of reagent into each tube and then pipet another 500 ul of reagent into each tube for a total of 1500u ul or 1.5 mL.
  5. Invert the tubes several times and then incubate at room temperatire (RT) for 10mins.
  6. Mix the 'blank' via pippeting and transfer to a 1000ul to a plastic, disposable cuvette.
  7. Zero the spectrophotometer using your blank sample. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  8. Mix the 'sample' via pippeting and transfer 1000 ul to a plastic, disposable cuvette
  9. Measure the absorbance at 595nm and record the value. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  10. . Remove the cuvette from the spectrophotometer. Using a P1000 set to 1000 ul, carefully pipette the solution in the cuvette up and down a couple of times to mix.
  11. . Measure the absorbance at 595nm and record the value a second time.
  12. . Average the two absorbance values you recorded.
  13. . Back-calculate your protein concentration using the standard curve below. Hint: Use the equation on the graph provided. The relationship between absorbance and concentration is linear and defined by the equation y=mx+b. You have the average absorbance of your sample, x, and you want to calculate the concentration, y. Don't forget to account for the dilution in step 2!
  14. . Give your protein sample to the TA for storage at -20ºC.

Standard curve was generated per Manufacturers Instructions . Please Read and Understand!

Pierce:Coomassie (Bradford) Protein Assay Kit

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