Lab 3: PCR
Objective:
Last time we extracted DNA from our soil samples. Today, we will use these samples as
templates for amplifying the 16S rRNA genes of bacteria by the polymerase chain
reaction (PCR). Specific primers for the bacterial 16S rRNA gens will be used (primers:
8F and 1492R). If you are unfamiliar with PCR, please review the following diagram.
You may also refer to one of your biology textbooks for further detail.
PCR:
5
3’
3
5’
’
Piece of DNA to be amplified
(fragment of chromosome)
’
5
’
3
’
3
’
5
’
5
5’
3’
’
5
’
5
3’
’
5
’3
5’
3’
’
3
’
3
’
53
’’
Heating to 94oC
Product Denaturation
DNA becomes single stranded
Cooling to 55oC
Primer Annealing
(8F and 1492R primers for
16S rRNA gene in bacteria)
5
’
53
’’
Heating to 72oC
Extension of DNA
by Taq polymerase and dNTPs
5
’
3
5’
Completion of one cycle
Two pieces of DNA from one
3’
5’
’
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16S rRNA:
Fundamental to molecular techniques has been the comparative analysis of
ribosomal ribonucleic acids (rRNA) sequences. This comparative study uses rRNA as a
marker because of its presence in all known organisms as a dominant cellular
macromolecule (Amann et al. 1995; Muyzer and Ramsing 1996; Amann 2000).
The existence of highly conserved and variable regions of rRNA sequences make
it an ideal candidate to use for the design of (PCR) primers to target general and specific
organisms. Finally, rRNA genes are not horizontally transferred among organisms
making this marker a unique “fingerprint” for a given organism (Amann et al. 1995;
Muyzer and Ramsing 1996; Amann 2000).
The rRNA molecule has three subunits: the 5S, 16S, and 23S in ascending order
by size. In recent years, the 16S molecule with its 1500 nucleotides of sequence
information has emerged as the yardstick for identifying bacteria and determining
phylogenetic relationships among different organisms (Muyzer and Ramsing 1996).
Although the 23S rRNA molecule has the greatest amount of information (3000
nucleotides) of the three molecules, current sequence databases for the 16S rDNA genes
are much larger than those of the 23S, which allows for better comparisons among
organisms. This remains true despite the fact that in some cases the 16S rRNA is
considered too well conserved to discriminate between organisms that are highly related
(Amann and Ludwig 2000). At present the 16S approach remains the “gold standard” for
explaining bacterial phylogeny (Ludwig and Schleifer 1999).
DN
A
23
S 5
16S
S
Ribosomes: sites for protein
synthesis; consist of RNA and
proteins
Three RNA molecules in
bacteria: mRNA, tRNA, rRNA
Three kinds of rRNA: 5S,16S,
23S
Genomic DNA (represented by closed loop on left) ~4720 kilo
bases (kb)
For 16S rRNA classification, only 1.5 kb of this DNA is
amplified
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Pre-lab Exercise 3:
Carefully read through Lab 3 and Laboratory Exercise 3 and answer the following
questions:
1. What happens during each temperature step in PCR?
2. What is considered to be the “gold standard” in bacterial phylogeny?
3. How large (kilo bases) is the 16S rRNA gene?
4. The 8F and 1492R primers amplify which gene?
5. What are the positive and negative controls here and how do they help you
interpret your results?
6. Using the table in the procedures as example, do the math required for 7
(25 l) reactions (not including the controls or losses to the tube). Fill out
the following table:
Reactions WITHOUT Betaine
Component
1x (25 l)
17.75 l
WATER
2.5 l
10X buffer w/
MgCl2
dNTPs
8F primer
1492R primer
BSA
Taq polymerase
Semi-total
Template
Total
0.5 l
0.5 l
0.5 l
2.0 l
0.25 l
24.0 l
1.0 l
25.0 l
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Laboratory Exercise 3:
Supplies (per person unless otherwise stated):
 Bunsen burner
 Ethanol spray bottle
 Paper towels
 Kim wipes
 Flint
 Waste container
 Laboratory tape
 Laboratory marker
 Ethanol resistant marker
 Bucket of ice
 Microcentrifuge (1 for doing PCR strip tubes)
 Thin walled PCR tubes (0.2 ml)
 Microcentrifuge tubes 0.7 ml (1 box of 50 per person)
 Pippettes, 0.5-10 l, 5-50l, 20-200l, 100-1000l (1 set per 2 persons)
 Pippette tips for all volumes
 Eppendorf Mastercycler Gradient PCR Machine (2 for the entire class)
 Freezer box
 Microwave
 250 ml pyrex bottles
 Gel casting trays and combs (for 5 lanes each) for each student
 Gel box
 Gel power source
 Small Tupperware tray
 Gel imaging system with printer and disk drive
 Saran wrap
Solutions:
 PCR core kit reagents
 8F and 1492R primers
 Betaine
 BSA
4
PCR Protocol:
___ 1. Remove the following PCR reagents from the freezer and thaw on ice.
i. Water (this is ultra pure sterile water)
ii. 10X buffer w/ MgCl2
iii. dNTPs
iv. 8F primer
v. 1492R primer
vi. BSA
vii. Betaine
viii. Positive Control
___ 2. Remove your DNA samples from last time and thaw on ice.
___ 3. You will be doing four reactions of 25 l each according to the table that
follows. Since there is always some loss of liquid in the pipetting and to
the tube, you will prepare for 7 reactions.
Reactions WITH Betaine
Component
WATER
1x (25 l)
12.75 l
7 x (25 l)
89.25l
2.5 l
0.5 l
0.5 l
0.5 l
2.0 l
5.0 l
0.25 l
24.0 l
1.0 l
25.0 l
17.5 l
3.5 l
3.5 l
3.5 l
14.0 l
35.0 l
1.75 l
168.0 l
(7 x 1.0 l)
175.0 l
10X buffer w/ MgCl2
dNTPs
8F primer
1492R primer
BSA
Betaine
Taq polymerase
Semi-total
Template
Total
___ 4. Prepare one master mix solutions (approx. 168 l in volume) according
to the table above but do not add the template! Take a sterile 700 ml
tube and begin adding each of the components listed in the table in the
order they are listed. STOP when you get to the template… you have
more than one template!!! Make certain than you insert the pipette tip
into the liquid completely when pipetting, you will be working with
small volumes at times!
___ 5. Dispense 24 l of the master mix solution you just prepared (without
template) to thin walled PCR tubes and number and label your tubes
with your information as below. Your reactions will include your
samples as well as positive (bacterial DNA) and a negative (pure water)
controls (YOUR STERILE TECHNIQUE IS VERY IMPORTANT
HERE!!! DO NOT CONTAMINATE YOUR TUBES):
Sample Label
1-your initials
2-your initials
3-your initials
4-your initials
5-your initials
6-your initials
Sample Information
Burn area 10 cm
Burn area 30 cm
Control area 10 cm
Control area 30 cm
Positive control
Negative control
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___ 6. Place your tubes on ice and wait for your classmates. Your samples will
be loaded onto a Mastercycler Gradient PCR Machine running the
following cycle. Upon completion, the samples will be refrigerated until
next class.
1.
2.
3.
4.
5.
6.
7.
Lid 105 oC
94 oC
94 oC
55 oC
72 oC
Goto Step #2
72 oC
4 oC
Wait
3 min
1 min
1 min
1 min
Repeat 29 times
10 min
Hold, enter
___ 7. Return all your reagents and samples to the appropriate freezer box.
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Laboratory Report #3:
This lab report will be combined with #4.
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