Molecular & Cell Biology Laboratory
Instructor: Elmar Schmid, Ph.D.
PCR amplification of bacterial genes
The protocol below allows the amplification of bacterial genes, e.g. for 16S-rRNA, from
isolated bacterial chromosomal DNA. Even though this protocol has proven to be
successful to amplify different bacterial genes (Pietsch A. & Schmid E., 2001), it may
require modifications, e.g. changes in the free Mg2+ concentration or the optimization
of the annealing temperature TA, to achieve optimum results.
Necessary Equipment:
-
GeneMate thermal cycler
Micro-centrifuge
(with rotor for 1.5 ml reaction tubes)
Adjustable-volume digital pipettes
(e.g. Eppendorf, Finnipette)
Plastic racks (for 0.2 ml reaction tubes)
Ice bucket (filled with crushed ice)
Pre-chilled Bench-top cooler
(e.g. “Strata-cooler”: Stratagene)
Required Materials & Reagents:
-
-
-
DNAse-free, sterile double-distilled water (= ddH2O) (Sigma)
Purified genomic bacterial (= template) DNA solution
(Stock solution: 1 μg/ml)
 genomic DNA isolated from inoculated bacterial colonies
Bacterial gene -specific forward & reverse primer pair (Stock solution: 5μM)
 designed and tested in a separate bioinformatics part of this Molecular
Genetics lab
Ready-To-GoTM PCR Beads (0.2 ml; Amersham: Cat # 27-9556-01)
dNTP solution (Working stock: 10mM)
Sterile plastic reaction tubes (1.5 ml; autoclaved, PP)
Sterile pipette tips (yellow, blue)
Sterile Crystal tips for PCR (with aerosol filters)
Vinyl or Latex gloves
Permanent marker pen
1
Molecular & Cell Biology Laboratory
Instructor: Elmar Schmid, Ph.D.
Procedure:
a.
Take following stock solutions out of the freezer and slowly thaw them
ahead of time; 10 – 15 min before starting with the reaction mixes
forward and reverse primer stock solutions (Stock: 5μM)
 your lab instructor will tell you which primer pairs you will be using
25mM MgCl2 solution
Purified genomic bacterial (= template) DNA solution (10 5 – 106 target
molecules)
 DNA Stock: ~ 1μg/ml
10mM dNTP solution (Optional)
b.
After the solutions have been thawed, place them into the Strata-Cooler
or (alternatively) into a metal rack placed into crushed ice
c.
Place lid-closed “Ready-to-Go” PCR reaction tubes (0.2 ml) in fitting
plastic racks on your bench and number them on the lids using a
permanent marker pen
d.
Pipette following solutions and components into the reaction tubes
following the pipetting scheme below; use fine-pointed, sterile Crystaltips
 wear vinyl gloves
 make sure you follow the sequence from top to bottom
 make sure to change the sterile crystal-tips between each
new pipetting step!
PCR reactions: Pipetting scheme
Bacterial DNA
Code+
Reaction tube #
ddH2O
25 mM MgCl2
5 μM fw-primer
5 μM rv-primer
Template DNA*
Total Volume [ μl ]
+
*
-
1
2
3
1
2
3
4
5
6
7
8
9
10
25
-
24
1
21.5
1.25
1.25
1
19
2.5
1.25
1.25
1
24
1
21.5
1.25
1.25
1
19
2.5
1.25
1.25
1
24
1
21.5
1.25
1.25
1
19
2.5
1.25
1.25
1
25
25
25
25
25
25
25
25
25
25
your instructor will give you the code for the used bacterial DNA
(1  3)
columns 2 – 4 will have the same bacterial genomic DNA (= DNA 1);
columns 5 – 7 will have the same bacterial genomic DNA (= DNA 2)
columns 8 – 10 will have the same bacterial genomic DNA (= DNA 3)
If you do PCR with genomic DNA from more than 3 different bacteria prepare and
set up correspondingly more PCR reaction tubes !
2
Molecular & Cell Biology Laboratory
Instructor: Elmar Schmid, Ph.D.
e.
Close the tubes and mix the contents by gently flicking the tubes with
your fingers
 make sure that all reaction components are at the bottom of
the PCR tube
f.
Briefly spin in a microfuge for a couple of seconds at high speed to
collect all the liquids at the bottom of the tubes
g.
Place all the reaction tubes into 96-well metal heating block of the
GeneMate thermocycler, close the reaction chamber with the heated lid
and perform the polymerase chain reactions using following cycle
conditions (= temperature program)
 your instructor will show you how to set up and program
the GeneMate thermal cycler
 make sure the machine runs with the “heated lid-ON” mode
PCR Program
1 Cycle (1x)
5 min
30 Cycles (30x)
1 min
1 min
2 min
1 Cycle (1x)
3 min
overnight
Temperature
94oC
Step
Denaturation
94oC
(Tm – 3)oC
72oC
Denaturation
Annealing
Extension
72oC
4oC
Final Extension
Sample Hold
Calculations: Melting ( Tm )and Annealing Temperature for the used Primer Pairs
 write the sequences of the primer pairs you are using into the table below
 calculate the temperatures based on your known primer sequences and using the
calculation formulas I or II above and write them into the table below
Primer
name
Primer sequence (5’  3’)
Tm
TAnneal
(calculated)
PQ. 1: Based on your calculations, which annealing temperature Ta are you using for
your PCR experiment? Argue.
Chosen Annealing Temperature:
T a = _____ oC
____________________________________________________________________
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Molecular & Cell Biology Laboratory
Instructor: Elmar Schmid, Ph.D.
h.
After you started the PCR reaction, return the used solutions and stocks
(primers, genomic DNA, dNTPs, etc.) back into the freezer to avoid
degradation
i.
After completion of the cycling program, turn off the machine and take
out all samples
 Careful: the heated lid might still be hot!
j.
Use the samples for subsequent gel electrophoresis, DNA visualization
and gel purification ( see separate lab manual) or store them in a
freezer at -20oC for later use
k.
Carefully clean the wells of the heating block from eventual
condensation water or spills using a soft paper tissue to avoid
corrosion damage to this expensive part of the thermal cycler
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