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Day 4: PCR amplification

Assignments Due

Lecture Topics

Overview of Experiment

In today's lab, you will set up several types of PCR reactions: the positive control ensures that the Taq DNA polymerase and the kit components are functional; the negative control demonstrates that in the absence of a specific template DNA, the primers alone do not amplify a specific product; the reactions using plasmid DNA are for amplification of specific BioBricks; colony PCR is used to screen putative recombinants after ligation/transformation.


Polymerase Chain Reaction

Polymerase chain reaction (PCR®) produces many copies of segments of DNA. The reproduction relies on the ability of DNA polymerase to make double stranded DNA. If a shorter, complementary segment of single stranded DNA (primer) is annealed to a longer single strand of DNA (template), the polymerase will extend the 3' end of the shorter segment to create double stranded DNA. Using two primers that are complementary to opposite strands, the DNA between and including the two primers is amplified. One cycle of amplification of DNA consists of three steps:

  1. Denature the double strands by melting (90-94°C)
  2. Anneal the primers to the template DNA at a reduced temperature (55-70°C)
  3. Synthesize (extend) the complementary DNA (70-75°C) with a DNA polymerase

Usually, 30 cycles are run yielding a near doubling of the number of DNA copies in each cycle. Actual efficiencies approach 1.8 fold increase in copy number/cycle.

Originally, DNA polymerase had to be added after each denaturation step but a thermal stable polymerase isolated from Thermus aquaticus (Taq) eliminated this tedious and expensive step. DNA fragments of fewer than 10,000 base pairs can be amplified with Taq. Longer fragments tend to fail due to mismatch errors that stall the enzyme. Several other thermal stable enzymes have been isolated that can survive the heating cycles. Each has varying efficiencies of proofreading and exonuclease activity for the correction of mismatch errors. A combination of two enzymes achieves amplification of longer lengths of DNA, up to 30-40 kb.

The efficiency of the PCR reaction necessitates scrupulous attention to keeping solutions clean and free from contamination. Some laboratories devote separate areas and equipment as PCR positive and PCR negative to prevent possible cross contamination. The preparation of target DNA should never occur in the same space as the preparation of the PCR reaction. (This guideline is not possible in the teaching lab making the negative control very important in our experiment.) Positive displacement pipettes or special filtered pipette tips are used to prevent contamination. For diagnostic and very sensitive procedures, reactions are set up in a laminar flow hood and all non-DNA solutions and containers are exposed to UV light to destroy errant DNA. Care must be taken to prevent contamination of the stock solutions. PCR on 1 µl of a 1:100 dilution of the plasmid preparation in this experiment can produce a visible band on the agarose gel.

The manufacturer's protocol recommends that 1 x 10(5)-1 x 10(6) target molecules be placed in the reaction mix. Consider the following relationships as a guideline for amplification of single copy genes:

Primer design is a critical component of PCR -- ideally, a primer pair will anneal specifically to the target sequence and amplify just that region. Online tools, such as Primer3 (Center for Genome Research, Whitehead Institute for Biomedical Research), and software packages, such as the Wisconsin Package from Genetics Computer Group (GCG), help you design effective primers for PCR. However, there are several simple guidelines you can follow to design good primers "by hand."

For some useful information, see these guides from Invitrogen and Promega.
***Just for fun, check out Bio-Rad's Scientists for Better PCR***


Experimental Procedures

A. Preparing bacterial colonies

PCR of an individual bacterial colony is a quick and relatively easy method to screen transformants. We use forward and reverse primers that bind upstream and downstream, respectively, of the multiple cloning site (MCS) on BioBricks or an internal primer (i.e., one that anneals to the insert DNA) with one of the MCS primers. The size of the product generated varies with the insert present in the BioBrick. Thus, any single colony producing an amplified fragment of the expected size is likely to contain the desired plasmid DNA.
  1. For each colony you pick, put 39 µl nuclease-free water in a sterile PCR tube containing a TaqBead™ (you will pick 4 colonies today)

    Other than the presence of colonies, is there any indication that your ligation/transformation was successful?

  2. Set pipette to 3 µl and use a sterile tip to lightly touch a single colony (do not remove ALL of the colony or gouge the agar!)
    Assign an identification symbol to each colony and label the bottom of the plate under the "spot" -- this plate can be incubated at 37°C for outgrowth of the individual colonies; you can culture any "positives" from this plate
  3. Gently pipet up and down to mix cells with water (make sure cells are well-mixed) but do not disturb the bead
  4. Proceed to Setting up PCR reactions

B. Setting up PCR reactions


Table 1: Construction of PCR Reaction Solutions

PCR Reactions:

Positive Control
(Promega Kit)

Negative Control
(no DNA)

Plasmid DNA

Bacterial
Colony

TaqBead™
polymerase

one bead

one bead

one bead

one bead

10X reaction buffer
(with MgCl2)

5 µl

5 µl

5 µl

5 µl

nuclease-free
water
(NF H2O)

36.4 µl

38 µl

38 µl

39 µl

nucleotide mix (dNTPs)
(10 mM)

1 µl

1 µl

1 µl

1 µl

primer 1

3.3 µl
C1

2.5 µl
VF2

2.5 µl
VF2

2.5 µl
VF2

primer 2

3.3 µl
C2

2.5 µl
VR

2.5 µl
VR

2.5 µl
VR

DNA template

1 µl
Control DNA (C)

1 µl
nuclease-free water

1 µl
plasmid DNA (P)
(1:100)

"touch"
bacterial
colony (C)

  1. Add reagents in the order given in the table (Note: DNA template for the bacterial colony reactions was added when you "touched" the colony with a pipet tip in Part A)
  2. Gently tap tube to mix.
  3. Take your samples to a thermal cycler (in B05-C) preheated to 95°C.
    NOTE: there are only 3 machines; the cycler will be started by the instructor when enough students are ready.
    Be certain to record in your notebook the position and labels of your samples and an I.D. of the instrument used.
  4. Cycling Conditions:

C. Agarose gel analysis of PCR products

  1. Prepare a medium 1% agarose gel with a 20-well comb (use ~120 ml molten agarose) as on Day 1 [pour one gel per team]
    NOTE: IF the PCR is not finished until 5:30 p.m. or later, you will analyze the reaction products at the beginning of lab day 5; remove the comb from your gel, cover the gel/gel tray with plastic wrap, and store gel at 4°C
  2. Preparation of PCR samples:
    a. Spot 2 µl 6X loading buffer (LB) on a piece of parafilm (one spot for each PCR sample)
    b. Using a sterile pipet tip, make a hole in the center of the paraffin wax overlay; use a fresh tip for each tube
    c. Insert a fresh tip through the hole and remove 10 µl from the 1st PCR tube, mix it with one spot of 6X LB by pipetting up and down, and load the sample into the well
    d. Repeat procedure for each PCR reaction
  3. Load 10 µl NEB Quick-Load 1 kb DNA Ladder
  4. Run the gel at 130 V for 20 minutes
  5. Photograph the gel and compare the observed bands to the standards
    • Did you get the expected size products?
    • Estimate the PCR yield by comparing the intensity of ethidium bromide staining of the products to the standards

Efficiency of Transformation (EOT) = # colonies / per µg of DNA
EOT is calculated by counting the number of colonies that grow on selective media following transformation and dividing by the total µg DNA used in the transformation (assume you used 0.05 µg DNA in the ligation reaction). Dilutions must be calculated to determine the amount of DNA present in the volume of transformed culture placed on each plate.

While your PCR reactions are running, count the colonies present on each of your plates to determine an average EOT for your procedure. If only a few colonies are present, count the entire plate. If many colonies are visible, place the plate on a grid such as a page of your notebook and count the number of colonies in four or five grids representing an average density across the plate. The rule in grid counting is to score any colonies in contact with the lines to the top and right side of the square but not those in contact with the other sides. Average the scores and multiply by the total area of the plate to calculate the total number of colonies.


Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 6 November 2009