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:

Titanium Taq Reaction Mix / Volume / Final Conc
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 / Volume / Final Conc
Titanium Taq PCR Buffer, 10X / 5 µl / 1X
Control Primer Mix / 1µl / 0.4 µM
Control Template / 1µl / 0.4 µM
50 X dNTP Mix (10 mM each) / 1µl
Nuclease-Free Water / 41 µl / N.A.
Taq Polymerase (50X) / 1µl / 1X

Negative control Rxn X 1:

Titanium Taq Reaction Mix / Volume / Final Conc
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
50 X dNTP Mix (10 mM each) / 1µl
Nuclease-Free Water / 41 µl / N.A.
Taq Polymerase (50X) / 1µl


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.