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Day 4: Isothermal assembly of vectors

Assignments Due

Lecture Topics

Overview of Experiment

In today's lab, you analyze the PCR reactions for the colony screen and the fragment for assembly on an agarose gel. After confirming that you have product for amplification of the kanamycin resistance cassette, you clean-up the PCR product and perform isothermal assembly.

Agarose gel analysis of PCR products

  1. Prepare a mini 1% agarose gel with two 8-well combs (use ~40 ml molten agarose) as on Day 2 [pour one gel per pair]
  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 in the PCR tube; 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
Expected products:

Purification of PCR Fragment for Assembly

After confirming that you have product, clean-up PCR reaction with a DNA Clean & Concentrator-5™ Kit (Zymo Research Corp., Orange, CA); elute DNA in 10 µl 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA.


Estimation of DNA Amounts by Intensity of Ethidium Bromide Fluorescence

If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA present can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA.

You need to estimate concentration of the purified and concentrated PCR fragment and prepared vector for isothermal assembly.  Compare the intensity of the "unknown" DNA (e.g., 2X as bright) to a band in the 1 kb ladder to estimate the DNA concentration of your sample (ng of DNA for each size standard are given in the Table on the manufacturer's insert).
  1. Preparation of samples:
    a. Put 2 µl of each DNA sample in a tube
    b. Add 8 µl NF-water
    c. Add 2 µl 6X LB
  2. Run an agarose gel comparing your "unknown" to a 1 kb ladder
  3. Photograph the gel
  4. Compare the relative intensity of staining of the unknown with a band in the 1 kb ladder
  5. Estimate the DNA concentration of your original sample (you need to consider the initial volume that contained the DNA; the volume loaded onto the gel is not critical to your calculation of concentration)

Isothermal Assembly: Gibson Method

Perform isothermal assembly (ITA) of the PCR product containing the kanamycin gene from pET-24d and the pET-21d vector that was digested with NcoI and treated with mung bean nuclease. Refer to the protocol written by your team for contents of the assembly master mix--make sure the components of the isothermal reaction buffer and the names/concentrations of each enzyme are recorded in your lab notebook.
  1. Mix equimass of each piece of DNA (50 ng each) - must be in 5 µl total volume
  2. Add 15 µl of 1.33x Assembly Master Mix (ITA) to DNA
  3. Incubate at 50°C for 60 min
  4. Clean-up 5 µl of ITA reaction with a DNA Clean & Concentrator-5™ Kit (Zymo Research Corp., Orange, CA)
    a. elute DNA in 5 µl 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA
    b. store at -20°C for transformation in the next lab
  5. Store 15 µl of ITA reaction at -20°C for agarose gel analysis in the next lab

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 13 October 2011