BIO/CHEM 475 Spring 2004

BIO/CHEM 475 Spring 2005
Week 2 (Young)
Master Mixes contain all of the ingredients needed to perform PCR (Taq polymerase may or
may not be part of the Master Mix). PCR is generally performed at 50 l per reaction. It is
possible to use as little as 25 or 15 l to save on cost of reagents.
The Master Mix buffer is often stored as a 10X stock solution (100 mM Tris-HCL, pH 8.3, 500
mM KCL, wetting agents, others) that is diluted to 1X for use. MgCl2 (0 – 4 mM) is stored as 25
mM stock. Store 10X buffer, MgCl2, deoxynucleotides, primers and Taq DNA polymerase
enzyme at -20oC.
MgCl2 complexes with dNTPS, primers and templates. Additionally, the final
concentration can be greatly influenced by chelating factors in storage buffers and DNA
extraction protocols. MgCl2 concentration may need to be determined empirically (1.5 –
2.5 mM is a good starting estimate).
To know / To do:
- Pipetters are not very accurate, or very consistent. One should avoid mixing single PCR
reactions in order to avoid using small volumes.
- Always mix (n + 1) master mix than is necessary, n being the number of reactions you plan on
- Regardless of the total volume, be certain to keep the final concentrations of reagents constant.
- It is easy to figure out how much stock reagent to use by following a simple formula:
(initial concentration) X ( volume needed )
(final concentration) X (volume of sample)
Primers are short strands of DNA (oligonucleotides) that are designed to be complementary to
sections of DNA targeted for amplification. Primers are annealed to the denatured DNA template
to provide an initiation site for the elongation of a new DNA molecule.
PCR primer design is an inexact science. Ultimately, primer function is determined empirically.
Once designed, primers are synthesized by companies eager to retain your patronage. Currently
(2004), a typical price is $0.75 per base. Turnaround time is generally 2-3 days.
To know / To do:
- Primers are generally used at 0.1 – 1.0 M. We will start with 0.2 M in our reactions.
- Primers arrive from the company dry, and need to be put into solution…
- for 50 l reactions…
 500X stock solutions are generally used,
 often in TE (10 mM Tris-Cl, 1 mM EDTA, pH 7.5 –8.0),
 working stocks (50X) are often made with RNase/DNase free ddH2O,
What would you do with 30 nmol of primer? Hint: start with a 500x stock.
Hint #2: so that 1 µl provides 0.2 µM
To make a 500X stock…
1. 100µM solution using 30 nmols of oligo:
0.03 µmol
1000 ml
x = 0.3 ml TE.
2. Make a 1X stock [9X ddH2O, 1X (10X stock)].
- Tm (melting temperature): theoretical temperature at which 50% of the oligo population is
annealed to the template target. A variety of formulas have been used for predicting Tm values,
none are accurate. Here is an example…
Estimation for Oligonucleotides: The melting temperature of an oligonucleotide is dependent
upon the length of the sequence, the G+C content of the sequence, and the type and concentration
of cation present, particularly sodium ion, Na+. The following formula is recommended for
oligonucleotides ranging in length from 20 to 100 residues, and [Na+] concentrations ranging
from 0.01 M to 1.0 M:
Tm = 81.5 + 16.6 log [Na+] + 41(G + C)/length - 500/length
For example, for a 40mer with a G+C content of 22 in a 0.1M NaCl solution:
Tm = 81.5 + 16.6 log [0.1] + 41(22)/40 - 500/40
Tm = 81.5 - 16.6 + 22.55 - 12.5
Tm = 74.95 ~ 75° C
Tm = 81.5 - 16.6 + 22.55 - 12.5
Tm = 74.95 ~ 75° C
Typical Master Mix (add reagents in order, and on ice)
ddH2O rnase/dnase Free
10x PCR Reaction Buffer
MgCl2 (final 1.5 mM)
dNTPS (final 0.2 mM each, 0.8 mM total)
Forward Primer (10 pmol/l)
Reverse Primer (10 pmol/l)
DNA (10pg-1µg)
in solution
Taq enzyme (add to master mix, or add to hot reaction)