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Day 4: Isothermal assembly of vectors
Assignments Due
- "Sniffers, buzzers, toggles and blinkers:
dynamics of regulatory and signaling pathways in the cell"
(Curr
Opin Cell Biol 2003,15:221-231)
- "Quantitative Modeling in Cell Biology: What Is
It Good for?" (Developmental Cell 2006, 11:279-287)
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
- 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]
- 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
- Load 10 µl NEB Quick-Load 1 kb DNA Ladder
- Run the gel at 130 V for 20 minutes
- 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:
Colony Screen
- Negative control = NO bands
- BBa_R0040 =
292 bp
- BBa_E08400 =
1218 bp
- R0040+E0840 (colony PCR) = ~ 1200 bp
Fragments for Assembly Reaction
- kanamycin resistance cassette in pET24d = ~1000 bp
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).
- 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
- Run an agarose gel comparing your "unknown" to
a 1 kb ladder
- Photograph the gel
- Compare the relative intensity of staining of
the unknown with a band in the 1 kb ladder
- 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.
- Mix equimass of each piece of DNA (50 ng each) - must be in 5 µl total volume
- Add 15 µl of 1.33x Assembly Master Mix (ITA) to
DNA
- Incubate at 50°C for 60 min
- 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
- 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