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 lacZor lacZαprimary primersto 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 / 1x / 104x
dNTPs (2 mM) / 3.75 µl / 390 µl
MgSO4 (25 mM) / 1.00 µl / 104 µl
DMSO / 1.25 µl / 130 µl
10% Sterile Glycerol / 14.50 µl / 1508 µl
10x KOD Buffer (provided with enzyme) / 2.50 µl / 260 µl
KOD Polymerase (1 Unit/µl) / 0.50 µl / 52 µl
Sm1021genomic DNA (100 ng/µl) / 0.50 µl / 52 µl
Primary Forward Primer (1.25µM) / 0.50µl
Primary Reverse Primer (1.25 µM) / 0.50 µl

1. 94°C for 2 min

2. 94°C for 1 min

3. 67°C for 1 min

4. 68°C for 6 min

5. 94°C for 1 min

6. 66°C for 1 min

7. 68°C for 6 min

8. 94°C for 1 min

9. 65°C for 1 min

10. 68°C for 6 min

11. 94°C for 1 min

12. 64°C for 1 min

13. 68°C for 6 min

14. 94°C for 1 min

15. 63°C for 1 min

16. 68°C for 6 min

17. 94°C for 1 min

18. 62°C for 1 min

19. 68°C for 6 min

20. 94°C for 1 min

21. 61°C for 1 min

22. 68°C for 6 min

23. 94°C for 1 min

24. 60°C for 1 min

25. 68°C for 6 min

26. Go to step 23 -12 times

27. 68°C for 6 min

28. 23°C for ever

29. end

Expected Run time: ~3 hrs 15 min

Secondary Cocktail / 1x / 104x
10% Sterile Glycerol / 3.55 µl / 369.2 µl
MgSO4 (25 mM) / 0.20 µl / 20.8 µl
DMSO / 0.25 µl / 26.0 µl
10x KOD Buffer (provided with enzyme) / 0.50 µl / 52.0 µl
Secondary Forward Primer (100 µM) / 0.25 µl / 26.0 µl
Secondary Reverse Primer (100 µM) / 0.25 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:

1.94ºC for 1 min

2.55ºC for 1 min

3.68ºC for 6 min

4.Goto step 1

5.Repeat steps 1–4 for 30 cycles

6.68ºC for 6 min

7.4ºC forever

Expected Run time: ~5hrs

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.