Lab 10/10/06: PLASMID ISOLATION/PCR ANALYSIS
The Polymerase Chain Reaction (PCR) is a procedure in which a target piece of
DNA is iteratively replicated resulting in the production of many copies. Its power, and
its peril, lies in its exquisite sensitivity; each PCR cycle theoretically doubles the amount
of targeted sequence in the reaction. Thus, 20 cycles increases the amount of target DNA
by a factor of more than a million in a matter of hours. This sensitivity allows the
detection of even the smallest amount of target (eg., a single-copy gene or low-copy
message), but it also allows the amplification of even the smallest amount of compatible
contaminants.
For today’s investigation, you will amplify an unknown target sequence in
plasmid DNA. You will start out by isolating plasmid DNA using a resin-based
procedure, the Wizard® Plus Minipreps DNA Purification System (protocol attached).
Following quantitation, you will dilute a sample of your plasmid to 50 ng/µl. This will
serve as your template stock for one PCR reaction. Additionally, you will test the
temperature on the accuracy of PCR running the reaction at 2 temperatures.
Plasmid Isolation Using the Wizard® Plus Minipreps DNA Purification
System
1. Grow 5 mls saturated overnight culture of E. coli in LB containing 50 µg/ml ampicillin
(from 50 mg/ml stock).
NOTE: BEFORE YOU BEGIN THE PLASMID ISOLATION TAKE THE
APPROPRIATE AMOUNT OF REAGENTS TO YOUR BENCH. USE REAGENTS
AT ROOM TEMPERATURE.
2. Pellet two 1.5 ml samples of saturated culture (3.0 mls total) by spinning @ top speed
in the µfuge for 1 m. Combine these samples by resuspending the combined pellets in
200 µl Cell Resuspension Solution (total).
3. Add 200 µl Cell Lysis Solution and mix by inverting the tube 4 times. NOTE: the cell
suspension should clear immediately.
4. Add 200 µl Neutralization Solution and mix by inverting the tube 4 times.
5. Pellet at top speed in µfuge X 5 m. Discard Pellet. If a pellet does not form, spin for
15 m more.
6. While pelleting, prepare a Wizard® Minicolumn for each prep as follows:
Remove the plunger from a 3 ml disposable syringe. Set aside.
Attach syringe barrel to the Luer-Lok® extension of the Minicolumn.
Pipet 1 ml of the resuspended resin into the barrel.
7. Carefully remove cleared lysate after pelleting (step 5), transfer to the barrel of the
Minicolumn/syringe assembly containing the resin.
8. Carefully insert the plunger into the syringe and gently push the slurry into the
Minicolumn.
9. IMPORTANT: detach the syringe from the minicolumn, THEN remove the plunger
from the syringe barrel. If you pull out the syringe with the MInicolumn attached it will
disrupt the resin bed.
10. Reattach the Minicolumn to the syringe.
11. Pipet 2 ml of Column Wash Solution into the barrel of the Minicolumn/syringe
assembly. Insert the plunger and gently push the Column Wash Solution through the
Minicolumn.
12. Detach the Minicolumn from the syringe, and place in a 1.5 ml centrifuge tube. Spin
at top speed for 2 m to drain the column.
13. Transfer Minicolumn to a new 1.5 ml centrifuge tube. Add 50 µl nuclease-free water,
wait 1 minute for the DNA to go into solution. Spin at top speed for 20 seconds to elute
the DNA.
14. Remove and discard the column. Quantitate spectrophotometrically (Dr. Davis will
demonstrate) @A260.
15. Dilute a portion of your plasmid isolation to 100 ng/µl. This will be used as the
template for the upcoming PCR reactions. Store the remainder of your mini-prep
sample at -20ºC. It will be used in later investigations.
Titanium Taq™ PCR Protocol
Taq DNA polymerase is a thermostable DNA polymerizing enzyme isolated from the
archaebacterium, Thermus aquaticus. Unlike most other DNA polymerases, it lacks the
3’to 5’ exonuclease activity, and thus is more error prone than other DNA pols. At its
optimum temperature 72ºC, Taq pol can amplify a 1 kb piece of DNA in ~30 seconds.
Another characteristic that has been exploited in molecular biology labs is its tendency to
add non-template directed adenines at the 3’ end of a strand. This is the basis for kits such
as the TOPO TA-cloning Kit ® (Invitrogen) which supplies vectors with compatible “T”
overhangs. Like other DNA pols, Mg+2 is essential for efficient catalysis. Other
necessary components for a PCR reaction are nucleotide triphosphates, template (target)
DNA, and primers. Primers are oligonucleotides that are complementary to the target
DNA. Like all DNA pols, Taq DNA polymerase can’t synthesize strands de novo; it can
only extend existing strands. The 10X buffer contains buffer and MgCl2.
Setting up the reactions (on ice) :
For a 50µl Rxn 1 X 2:
Volume
Final Conc
Titanium Taq Reaction Mix
Titanium Taq PCR Buffer, 10X 5 µl
1X
M13 Forward Primer, 10 µM
1µl
0.4 µM
M13 Reverse Primer, 10 µM
1µl
0.4 µM
DNA Template*
1µl
50 X dNTP Mix (10 mM each)
1µl
Nuclease-Free Water
40 µl
N.A.
Taq Polymerase (50X)
1µl
1X
* use 100 ng template for this reaction. Calculate concentration.
Positive Control Rxn X 1:
Titanium Taq Reaction Mix
Titanium Taq PCR Buffer, 10X
Control Primer Mix
Control Template
50 X dNTP Mix (10 mM each)
Nuclease-Free Water
Taq Polymerase (50X)
Volume
5 µl
1µl
1µl
1µl
41 µl
1µl
Final Conc
1X
0.4 µM
0.4 µM
Negative control Rxn X 1:
Titanium Taq Reaction Mix
Titanium Taq PCR Buffer, 10X
M13 Forward Primer, 10 µM
M13 Reverse Primer, 10 µM
50 X dNTP Mix (10 mM each)
Nuclease-Free Water
Taq Polymerase (50X)
Volume
5 µl
1µl
1µl
1µl
41 µl
1µl
Final Conc
1X
0.4 µM
0.4 µM
N.A.
1X
N.A.
Using the Thermocycler:
Our thermocyclers have a heated lid, so the reaction mixture doesn’t require
overlaying with mineral oil the reduce evaporation (Why is this an important
consideration?). The cycle you will be using is labeled LAB2, and consists of the
following steps:
Step 1- 95ºC for 5 m: Initial melting of template
Step 2-94ºC for 1 m: Denaturation
Step 3-52ºC for 1 m: Annealing
Step 4-72ºC for 1 m: Extension
Step 5- cycle through steps 2-4 X 30 cycles
Step 6-72ºC for 5 m: Clean-up extension
Step 7-4ºC until termination: storage
You may pick up your samples after step 6. Either store in the freezer or perform
gel electrophoresis.
For electrophoresis, load the entire reaction mixture on a 1% agarose gel and run
until the blue dye is about 1 cm from the bottom of the gel. For size determination, use
the 1kb ladder as a standard. Stain and photograph. Determine the size of the amplified
fragment. Be sure to include the PCR reaction conditions and the electrophoresis
conditions in your lab notebook.