Sensitivity of PCR II
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.
This week you will evaluate the sensitivity of PCR by using various dilutions of template. Starting with 100 ng of template, you will dilute the sample in 1:100 increments until you’re using 1 femtogram (10-15g) of template. For this exercise, you will make a master mix to facilitate handling smaller volumes (20 µl reactions instead of 50 µl reactions). Using your plasmid from Week 2, dilute it (if necessary) to 100 ng/µl. Dilute this sample 1:100 (i.e., 1 ng/µl). Perform a serial dilution, diluting each new sample 1:100 until you have diluted the original 100 ng/µl sample to 1fg/µl. You should now have template for 5 reactions.
Setting up the reactions (on ice) :
For a 20µl Rxn:
Titanium Taq Reaction Mix / Volume / Final ConcTitanium Taq PCR Buffer, 10X / 1X
M13 Forward Primer, 10 µM / 0.4 µl
M13 Reverse Primer, 10 µM / 0.4 µl
DNA Template or water / 0.4 µl / Variable
50 X dNTP Mix (10 mM each) / 0.4 µl
Taq Polymerase / 0.4 µl / N.A.
Nuclease-Free Water
FINAL VOL / 20 µl
Fill in the spaces in the table where appropriate.
NOTES: the primers are complementary to regions flanking the Multiple Cloning Site (MCS) in the plasmid.
Master Mix: Make up a master mix containing everything except template. In other words, you will make up a mix to which you will add template or water after distributing to separate tubes. Because the final volume will be 20µl, and you will add 1µl template, you must distribute 19µl of Master Mix/PCR tube. When making up the Master Mix aliquot the water first, enzyme last. Make up enough Master Mix for 7 reactions, then distribute. Complete the table prior to lab on Thursday.
Titanium Taq PCR Buffer, 10X / 14 µl
M13 Forward Primer, 10 µM / 2.8µl
M13 Reverse Primer, 10 µM / 2.8µl
50 X dNTP Mix (10 mM each) / 2.8µl
Taq Polymerase (50X) / 2.8µl
Nuclease-Free Water / 107.8
FINAL VOL / 133 µl
Positive Control Rxn X 1: Even though this is a 50 µl reaction, only load 20µl+dye.
Titanium Taq Reaction Mix / Volume / Final ConcTitanium Taq PCR Buffer, 10X / 5 µl
Control Primer Mix / 1µl
Control Template / 1µl
50 X dNTP Mix (10 mM each) / 1µl
Nuclease-Free Water / 41 µl
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, add 10 X loading dye and load the entire reaction mixture on a 1.2% agarose gel and run until the blue dye is about 1 cm from the bottom of the gel. Stain in EtBr as Project I, Module 5. 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, electrophoresis conditions, and staining/destaining conditions in your lab notebook.