Lab 3: Reverse transcription and end-point PCR
Lab Objectives
  • Reverse Transcribe RNA into complementary DNA (cDNA)
  • Amplify gene of interest using end point polymerase chain reactions (PCR)
  • Make an agarose gel




Reverse transcription
Supplies and Equipment:
  • Micropipettes (1-1000 μl)
  • Sterile filter pipette tips (1-1000 μl)
  • Tip waste jar
  • PCR tubes (0.5 ml; thin walled)
  • RNA samples (student provided)
  • M-MLV reverse transcriptase
  • M-MLV 5X reaction buffer
  • Oligo dT
  • dNTPs
  • Nuclease Free water
  • thermal cycler
  • microfuge tube racks
  • PCR tube racks
  • ice buckets
  • Kimwipes
  • Lab coat
  • Safety glasses
  • gloves

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 dapted from Wikipedia

REVERSE TRANSCRIPTION PROTOCOL
  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 last week)
    2. 1 μl of oligo dT
    3. 4 μl of nuclease free H2O
  3. Incubate the mixture for 5 min at 70C on the thermocycler then immediately transfer to ice. Briefly centrifuge you tube and the add the following:
    1. 5 μl of M-MLV 5X Reaction Buffer
    2. 5 ul of dNTPs
    3. 1 μl of M-MLV RT
    4. 4 μl of nuclease free H2O
  4. Incubate the mixture for 60 min at 42C and then heat inactivate at 70C for 3 min on the thermocycler.
  5. Spin down the sample in a desk top centrifuge.
  6. Store on ice or at -20C


Polymerase chain reaction
Supplies and Equipment:
  • Micropipettes (1-1000 μl)
  • Sterile filter pipette tips (1-1000 μl)
  • Tip waste jar
  • PCR tubes (0.5 ml; thin walled)
  • 1.5 ml microcentrifuge tubes (RNAse free)
  • cDNA (student provided)
  • dNTPs
  • 2x GoTaq Green Master Mix
  • Primers
  • Nuclease Free water
  • thermal cycler
  • Kimwipes
  • microfuge tube racks
  • PCR tube racks
  • ice buckets
  • Kimwipes
  • Lab coat
  • Safety glasses
  • Gloves


Procedure background
The polymerase chain reaction involves selective amplification of a DNA (genomic or complementary) target using the enzyme polymerase (after which the method is named), primers (short oligonucleotides), and dNTPs (A, C, T, and G). The method relies on thermal cycling, which consist of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. These heating and cooling cycles are comprised of three primary steps. A single cycle begins with denaturation at ~94C and during this step the DNA is melted or rather unwound and the strands are pulled apart resulting in single stranded DNA. This is followed by an annealing step at ~50-60C where the primers anneal to the target sequence. After which there is an extension step at ~72C where nucleotides DNA polymerase synthesizes a new DNA strand by adding dNTPs that are complementary to the template. As PCR progresses, the newly generated DNA is also used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. This process allows us to generate thousands to millions of copies of a particular DNA sequence with only a single or a few copies of a piece of DNA.

For this lab we will be using Promega’s Go-Taq green master mix , please read the manufacture’s protocol.

PCR PROTOCOL
You will be preparing four 50 ul reactions: two containing your cDNA as template and two containing no template, which are negative controls.

1. Make a reaction master in a 1.5 ml microcentrifuge tube labled "MM" and with your initials containing the following:
  • 250 ul of GoTaq®Green Master Mix, 2X
  • 15 ul of forward primer, 10 uM
  • 15ul of reverse primer, 10 uM
  • 108 ul of nuclease free H20
2. Pipette 48 ul of your master mix in to each of your 4 0.5 ml PCR tubes labeled 1 though 4 and with your initials
3. Add 2 ul of the appropriate template to each tube and mix via pipetting
Tube
Template
1
cDNA
2
cDNA
3
Nuclease-free H20
4
Nuclease-free H20
4. Spin tubes to pool liquid at the bottom of the tubes. Load reactions into thermocycler making sure the caps on tightly secured.
5. Your samples will be put through the following thermal cycling profile and then stored at -20C afterwards.
Step
Temperature
Time
Cycles
Denaturation
95C
5 min
1
Denaturation
95C
30 sec
40
Annealing
55C
30 sec
Extension
72C
90 sec
Final extension
72C
3 min
1
Hold
4C

1



Making an agarose gel

This gel will be used for agarose gel electropghoresis in lab next week.
Supplies and Equipment:

  • Micropipettes (1-1000 μl)
  • Sterile filter pipette tips (1-1000 μl)
  • Tip waste jar
  • 1L flask
  • agarose
  • 1X TAE
  • Ethidium bromide
  • Microwave
  • Gel rigs
  • Kimwipes
  • Lab coat
  • Safety glasses
  • gloves

AGAROSE GEL POURING PROCEDURE (groups of 4)
  1. Weigh 2g of agarose and mix with 150mL 1x TAE in a 1L flask
  2. Microwave solution for ~ 3 minutes
  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
  6. After gel is set, wrap in plastic wrap and place gel in the fridge for next week.