PCR amplification of the ORFeome
A major concern at the beginning of the project was that we would be unable to obtain consistent
PCR success using a high fidelity DNA polymerase since, because the primer sequences were to
be determined by the DNA sequences at the ends of the ORFs, there was no opportunity to
choose primer sequences that would optimize the PCR reaction. We therefore screened several
DNA polymerases and protocols for their ability to work with non-optimized primer sets and
with the relatively high (62%) G+C template. The touchdown PCR protocol described below
that uses a hot-start version of the high fidelity KOD DNA polymerase (Novagen, Inc., Madison,
WI link B2-and_F2_Novagen_PCR_guide.pdf) was far better than any other protocol we tried
with any of several DNA polymerases (link to Maloney et al. 2004 poster).
Set up for the Nested PCR Reactions
1. Remove all the PCR components from the freezer, place on ice and allow to thaw.
2. Aliquot 1 µl of a diluted primer set to a pre-labeled 0.2 ml tube
–record which primer set went into which tube
Primer stocks were prepared at a concentration of 100 pmole/µl (100 µM). Primers for
PCR were prepared by adding 1 µl each of the primary forward and the primary reverse
primer stocks to 78 µl of sterile distilled water to give a diluted primer stock
concentration of 1.25 µM for each primer.
3. Mix the PCR reaction cocktail (listed below)
4. Internal lacZ PCR amplification control. Before Sm1021 genomic DNA is added to the PCR
reaction cocktail, remove 23 µl and add it to the E. coli K-12 reaction control located in Strip
12 tube 8. Take a pre-aliquoted tube containing E. coli DNA and add 1 µl of the prediluted
lacZ or lacZα primary primers to the tube located in Strip 12 tube 8.
5. No DNA control. Before the Sm1021 genomic DNA is added to the PCR reaction cocktail,
remove 25 µl and add it to Strip 12 tube 7.
6. After the cocktail is mixed, aliquot 24 µl to each of the tubes using the Eppendorf
multichannel pipette. Remember not to add more cocktail to the controls located in Strip 12
tube 7 and Strip 12 tube 8.
Primary Cocktail
dNTPs (2 mM)
MgSO4 (25 mM)
DMSO
10% Sterile Glycerol
10x KOD Buffer (provided with enzyme)
KOD Polymerase (1 Unit/µl)
Sm1021genomic DNA (100 ng/µl)
Primary Forward Primer (1.25 µM)
Primary Reverse Primer (1.25 µM)
1x
3.75 µl
1.00 µl
1.25 µl
14.50 µl
2.50 µl
0.50 µl
0.50 µl
0.50µl
0.50 µl
104x
390 µl
104 µl
130 µl
1508 µl
260 µl
52 µl
52 µl
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
94°C for 2 min
94°C for 1 min
67°C for 1 min
68°C for 6 min
94°C for 1 min
66°C for 1 min
68°C for 6 min
94°C for 1 min
65°C for 1 min
68°C for 6 min
94°C for 1 min
64°C for 1 min
68°C for 6 min
94°C for 1 min
63°C for 1 min
68°C for 6 min
94°C for 1 min
62°C for 1 min
68°C for 6 min
94°C for 1 min
61°C for 1 min
68°C for 6 min
94°C for 1 min
60°C for 1 min
68°C for 6 min
Go to step 23 -12 times
68°C for 6 min
23°C for ever
end
Expected Run time: ~3 hrs 15 min
Secondary Cocktail
10% Sterile Glycerol
MgSO4 (25 mM)
DMSO
10x KOD Buffer (provided with enzyme)
Secondary Forward Primer (100 µM)
Secondary Reverse Primer (100 µM)
1x
104x
3.55 µl
0.20 µl
0.25 µl
0.50 µl
0.25 µl
0.25 µl
369.2 µl
20.8 µl
26.0 µl
52.0 µl
26.0 µl
26.0 µl
Transfer 65 µl per tube into 8 strip tubes and then aliquot 5 µl per tube into the 12 strips using an
8X multichannel pipettor. The quantities above were formulated to provide an extra 5 µl per row
in case additional material is needed.
PCR was continued by running the following program:
1.
2.
3.
4.
5.
6.
7.
94ºC for 1 min
55ºC for 1 min
68ºC for 6 min
Go to step 1
Repeat steps 1–4 for 30 cycles
68ºC for 6 min
4ºC forever
Expected Run time: ~5 hrs
S. meliloti genomic DNA was purified using QIAGEN’s DNeasy Tissue kit following the
manufacturer’s recommendations (QIAGEN Inc., Valencia, CA). Briefly, 1.5 ml aliquots of
bacterial cells from a 48 h culture of S. meliloti 1021 were harvested by centrifugation then
resuspended in lysis buffer. The genomic DNA was then bound to a silica-gel-membrane,
washed with provided buffers to remove salts and eluted from the membrane using 100 µl sterile
distilled H20. All the samples were pooled and diluted to 100 ng/µl, dispensed into 68 µl
aliquots, frozen and stored at -20°C.
We use 12 strips with 8 tubes each to represent a 96 well plate. To dispense the PCR cocktail to
the 12 strips, we first aliquot 305 µl into 8 strip tubes and then use an 8X multichannel pipettor
to aliquot 24 µl into each tube in the 12 strips. The quantities above were formulated to provide
an extra 24 µl per row in case additional material is needed.
PCR amplification was done in a 96 well format using an MJ Research PTC-200 DNA Engine
(MJ Research, Inc., San Francisco, CA) or similar machine. Amplification first used the primary,
ORF-specific, set of primers then the secondary primers were added to the reaction to provide
template for the attB sites. The following temperature cycling program was used. It includes a
hot start activation and a progressively decreasing set of annealing temperatures for touchdown
PCR during the initial phases of the reaction.