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Not everything that can be counted counts,
and not everything that counts can be counted.
Albert Einstein

Day 3: Polymerase Chain Reaction (PCR)

colony screen; fragment amplification for isothermal assembly


Assignments Due

Lecture Topics

Overview of Experiment

In today's lab, you set up several types of PCR reactions for the colony screen: 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. Additionally, you use PCR to amplify fragments for isothermal assembly.  Finally, you evaluate the transformations and calculate the efficiency of transformation.

Background


PCR Colony Screen

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 36 µl nuclease-free water in a sterile PCR tube containing a TaqBead™ (you will pick 3 colonies today)
  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:

Negative Control
(no DNA)

Plasmid DNA

Bacterial
Colony

TaqBead™
polymerase

one bead

one bead

one bead

10X reaction buffer
(with MgCl2)

5 µl

5 µl

5 µl

nuclease-free
water
(NF H2O)

38 µl

38 µl

36 µl
*add 1st

nucleotide mix (dNTPs)
(10 mM)

1 µl

1 µl

1 µl

primer 1

2.5 µl
VF2

2.5 µl
VF2

2.5 µl
VF2

primer 2

2.5 µl
VR

2.5 µl
VR

2.5 µl
VR

DNA template

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:

PCR of Fragments for Isothermal Assembly

  1. Add the reaction components in the following order:
  2. Cycling conditions:

Preparation of Vector for Assembly

  1. Digest 5 µl pET21 stock with NcoI: use NEB protocol & buffer for a total reaction volume of 50 µl
  2. Treat digested vector with mung bean nuclease: add 1 µl and incubate at 30°C for 1 hour
  3. Clean-up DNA with a DNA Clean & Concentrator-5™ Kit (Zymo Research Corp., Orange, CA); elute DNA in 5 µl 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA
  4. Store DNA at -20°C

Efficiency of Transformation (EOT)

EOT = # colony forming units / 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 50 ng DNA in the transformation reactions). If only a fraction of the transformed cells is used, dilutions must be taken into account to determine the amount of DNA present in the volume of transformed culture placed on each plate.

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 20 October 2011