SAN DIEGO MESA COLLEGE
Introduction to Microbiology Laboratory (Bio205)
Instructor: Elmar Schmid, Ph.D.
PCR amplification of bacterial 16S-rRNA gene
Laboratory Objectives
After completion of this lab you should:
1. have a deep understanding of the working principle of the polymerase chain
reaction (PCR) method
2. be able to name the reagents and components, including Taq polymerase,
primers, deoxynucleotide triphosphates (dNTPs) and DNA, necessary to
conduct a PCR experiment
3. know how to use a micropipettor to transfer micro-liter amounts of volumes;
understand the meaning of melting temperature (Tm) of a given PCR primer and
also be able to determine the annealing temperature (Ta) of a given PCR
experiment
4. have an understanding of the working principle of a thermal cycler (“PCR
machine”) and know how to professionally operate it
5. understand the importance of bacterial rRNA genes, especially the gene for
16S-rRNA, for phylogenetic analysis in modern microbiology
Necessary Equipment:
-
Thermal cycler (with heated lid)
Micro-centrifuge
(with rotor for 1.5 ml reaction tubes)
Adjustable-volume micropipettors
(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)
Template DNA solution (containing purified genomic DNA of Citrobacter
freundii and/or Enterobacter cloacae)
- this DNA stock solution is either supplied by the instructor or has been prepared
by the student in the previous lab session  see DNA Isolation Protocol)
16S-rRNA gene-specific forward/reverse primer pair (Stock solution: 10 μM)
 recognizing highly conserved gene regions of the C. freundii 16S rRNA gene
Ready-To-GoTM PCR beads (0.2 ml; Amersham: Cat # 27-9556-01)
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
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SAN DIEGO MESA COLLEGE
Introduction to Microbiology Laboratory (Bio205)
Instructor: Elmar Schmid, Ph.D.
Procedure:
Important: To avoid contamination with your own DNA or DNA degrading
enzymes on your hands, wear clean vinyl or latex gloves throughout all the
steps below!
a.
Take following stock solutions out of the freezer and slowly thaw the frozen
contents before starting with the PCR mixes
One forward (fw) and one reverse (rv) primer stock solution (5μM each)
 the primer pairs in use anneal with highly conserved regions of the
Citrobacter freundii (Cf) 16S-rRNA gene
 the primer pairs in use will lead to a PCR amplification product of
either 256 base pairs (fw1/rv1) or 700 base pairs (fw3/rv2)
- your instructor will tell you in the lab which primer pair your team will
be using!
Purified genomic (= template) DNA solution (105 – 106 target molecules)
 Citrobacter freundii (Cf) DNA (Stock: ~ 1μg/ml)
b.
After the solutions have been thawed, immediately place them on ice (in an
ice bucket)
c.
Place 2 closed “Ready-to-Go” PCR reaction tubes (0.2 ml) in plastic racks
on your bench and number them on the lids using a permanent marker pen
 make sure that the small whitish wax ball is visible at the bottom of the
reaction tube (sometimes it is stuck to the lid of the reaction tube!)
d.
Use a micropipettor and pipette following solutions and components into
the reaction tubes following the pipetting scheme below; use fine-pointed,
sterile Crystal-tips
 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
Reaction tube #
[ μl ]
ddH2O
fw-primer
rv-primer
Template DNA*
Total Volume [ μl ]
*
1
2
16
2
2
-
14
2
2
2
20
20
= Citrobacter freundii or Enterobacter cloacae DNA
What is the function of the reaction tube #1?
______________________________________________________________
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SAN DIEGO MESA COLLEGE
Introduction to Microbiology Laboratory (Bio205)
Instructor: Elmar Schmid, Ph.D.
Would you expect a PCR amplification product with sample #1? Why or
Why not?
____________________________________________________________
____________________________________________________________
Which reaction tube is supposed to have a PCR amplification product after
completion of the PCR?
____________________________________________________________
e.
Close the PCR reaction tubes and gently mix the contents by flicking the
tubes with your fingers
 make sure that all reaction components are at the bottom of
the PCR tube
 make sure that all tubes are clearly labeled on the lid and carry your/
your teams identification code!
f.
Briefly spin the PCR reaction tubes 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
thermocycler
h.
Your instructor will now program the thermal cycler using the PCR cycle
conditions (= temperature program) as shown in the Table below and then
start the polymerase chain reaction
PCR Program
1 Cycle (1x)
4 min
30 Cycles (35x)
0.5 min
0.5 min
2 min
1 Cycle (1x)
5 min
overnight
Temperature
95oC
Step
Denaturation
95oC
56oC
72oC
Denaturation
Annealing
Extension
72oC
4oC
Final Extension
Sample Hold
Calculations: Melting ( Tm ) and Annealing (TA) Temperature for the used Primer
Pairs
Write the sequences of the primer pairs you are using in the PCR lab into the table
below (your instructor will supply you with the DNA sequence of the primer in use!)
Calculate the temperatures based on your known primer sequences by using the
calculation formulas I and II shown in the PCR section of the Microbiology website
and write them into the table below
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SAN DIEGO MESA COLLEGE
Introduction to Microbiology Laboratory (Bio205)
Instructor: Elmar Schmid, Ph.D.
Primer
name
Primer sequence (5’  3’)
Tm
TAnneal
(calculated)
Based on your calculations, which annealing temperature Ta are you using for
your PCR experiment? Argue.
Chosen Annealing Temperature:
T a = _____ oC
____________________________________________________________________
i.
After you started the PCR reaction, return the used solutions and stocks (=
primers & genomic DNA) back into the freezer to avoid degradation
j.
After completion of the cycling program, turn off the machine and take out
all samples
 Careful: the heated lid might still be hot!
k.
Use the samples for subsequent agarose gel electrophoresis, DNA
visualization and gel purification ( see separate lab manual) or store them
in a freezer at -20oC for later use
l.
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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