Chapter 6
The Polymerase Incomplete Primer Extension (PIPE) Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis
Heath E. Klock and Scott A. Lesley
Summary
Significant innovations in molecular biology methods have vastly improved the speed and efficiency of traditional restriction site and ligase-based cloning strategies. "Enzyme-free" methods eliminate the need to incorporate constrained sequences or modify Polymerase Chain Reaction (PCR)-generated DNA fragment ends. The Polymerase Incomplete Primer Extension (PIPE) method further condenses cloning and mutagenesis to a very simple two-step protocol with complete design flexibility not possible using related strategies. With this protocol, all major cloning operations are achieved by transforming competent cells with PGR products immediately following amplification. Normal PCRs generate mixtures of incomplete extension products. Using simple primer design rules and PGR, short, overlapping sequences are introduced at the ends of these incomplete extension mixtures which allow complementary strands to anneal and produce hybrid vector/insert combinations. These hybrids are directly transformed into recipient cells without any post-PCR enzymatic manipulations. We have found this method to be very easy and fast as compared to other available methods while retaining high efficiencies. Using this approach, we have cloned thousands of genes in parallel using a minimum of effort. The method is robust and amenable to automation as only a few, simple processing steps are needed.
Key words: Cloning; Ligase independent; Enzyme free; Site-directed mutagenesis; PIPE; Incomplete primer extension
6.1. Introduction
Contemporary cloning strategies outline mainly iterative protocols based on ligase-independent methods (1-13). Most of these methods require specific sequences for successful cloning. Recombina-torial cloning is only one example where specific sequences must be incorporated and can encode extra, unwanted residues into expressed proteins (9). "Enzyme-free" cloning alleviates sequence
Sharon A. Doyle (ed.), Methods in Molecular Biology: High Throughput Protein Expression and Purification, vol. 498 © 2009 Humana Press, a part of Springer Science + Business Media, Totowa, NJ Book doi: 10.1007/978-1-59745-196-3
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requirements through a series of PCR steps and product treatments (10). Likewise, the PIPE method also eliminates sequence constraints, and it also reduces cloning and site mutagenesis to a single PCR step and transformation. These combined innovations make the PIPE method very fast, cost effective, and highly efficient. The following protocol includes all the wet lab steps, from making competent cells to submitting samples for sequencing, necessary for successful cloning and mutagenesis. Although the various examples presented here are shown with the expression vector pSpeedET (in-house), the PIPE method can be used with other vectors.
6.2. Materials
6.2.1. Preparation of Competent Cells
6.2.2. Preparation of Selective LB Agar Plates
6.2.3. PCRs for Amplifying Cloning Vectors and Inserts or Generating Site-Directed Mutants
1. Milli-Q water (see Note 1).
2. LB Broth: 25g of Difco" LB Broth (Miller) per 1 L of deion-ized (DI) water and autoclaved for 30min at 121°C. The media is autoclaved in 2,000 ml Kimax® Baffled Culture Flasks (ThermoFisher Scientific, Waltham, MA).
3. Sterile, disposable Corning Erlenmeyer polycarbonate flasks (500ml) (available through ThermoFisher Scientific).
4. MgCl2 Solution: 100 mM MgCL, in Milli-Q water, then sterile filtered. Stored at 4°C.
5. CaCl2 + Glycerol Solution: lOOmM CaCl2 and 15% glycerol in Milli-Q water, then sterile filtered. Stored at 4°C.
6. Microcentrifuge tubes (2 ml).
1. Sterile square BioAssay trays with 48-wcll dividers (Genetix, Boston, MA).
2. LB Agar: 40 g of Difco" LB Agar (Miller) per 1 L of DI water and autoclaved for 30min at 121°C.
3. Antibiotics (working concentration): Ampicillin (lOOug/ml), chloramphenicol (20ug/ml), kanamycin (30(Xg/ml).
1. Template DNA (20-100pg per PCR amplification).
i. For cloning vector amplifications (V-PIPE), use a recipient expression plasmid such as pSpeedET (see Fig. 6.1a).
ii. For insert amplifications (I-PIPE), use genomic DNA, cDNA, PCR product, or miniprepped DNA from a previously generated clone (see Fig. 6.1b).
iii. For mutagenic amplifications (M-PIPE), use the miniprepped DNA from a previously generated clone (see Fig. 6.1c).
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A. V-PIPE
r BAD/T7				
	MGSDKIHHHHHHENLYFOG	ccdB	STOP-cgcgac-Pac 1	
5' vector sequence: Primer 1 ->
3' - GTA GTAGTAGTAGTAGTG CTT TTG GAC ATG AAG GTC CCG-5'
Primer 2 -> 3' vector sequence:
^áTTAAA CGGTCTCCAG CTTGGCTGTT TTGGC-3' TAC CCG GGT AAG TTT AAA CGGTCTCCAG CTTGGCTGTT TTGGCGGATG AGAGAAGATT T
B. I-PIPE
Primer 3 -» Protein of Interest DNA sequence: Primer 4 >
GCA TGCGATGAA TTC GGG CAC ATA AAG-3'
TTG GGA GTC GCT AGT TGTCAT ACC ATG
C. M-PIPE
r BAD/T7	MGSDKIHHHHHHENLYFOG	POI("A ->Y")	STOP-cgcgac-Pac I			
						
				Kan '		
Substitutions (LMN ->GGG):
Primer 5 -> Protein of Interest sequence: Primer 6 ♦
3-ACG CTA CTT AAG CCC GTG TAT £
TCAG CGA TCA ACA GTA TGG TAG 3'
Deletions ( A LMN):
Primer 7 -> Protein of Interest sequence: Primer 8 ->
^^3jg CCG CAG SSA TCA ACA GTA TGG TAa KL MNPQRSTVWY 3' - ACG CTA CTT AAG CCC GTG TAT TTC GGCq^ 0
Insertions (M -» INS *r N): ^
Primer 9-» Protein Ol Interest sequence: Primer 10-»
CAAC CCT CAG CGA TCA ACA GTA TGG TAa G HIKLMNPQRSTVWY-3-ACG CTA CTT AAG CCC GTG TAT TTC CCT TAC
Fig. 6.1. Oligonucleotide design for the three common PCR amplifications used in the PIPE method. The 15 base complementary overlaps are shown as the underlined portion of each primer sequence, (a) V-PIPE (vector PCR). Primers 1 and 2 are examples of primers that could be used to PCR amplify a vector, such as speedet, in a way suitable for annealing to inserts amplified in (b). (b) l-PIPE (insert PCR). Primers 3 and 4 are examples of primers that could be used to PCR amplify inserts from various templates in a way suitable for annealing to the vector amplified in (a). PIPE cloning works by intermolecular annealing across the two annealing sites of the (a) and (b) PCRs. (c) M-PIPE (mutagenic PCR). Primers 5 and 6 represent primers which could be used to create substitution mutants. Primers 7 and 8 represent primers which could be used to create deletion mutants. Primers 9 and 10 represent primers which could be used to create insertion mutants. PIPE mutagenesis works by intramolecular annealing across the single site of a (c) PCR.
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2. Oligonucleotide primers: Ordered at 50 uM (Integrated DNA Technologies, Coralville, IA).
a) V-PIPE primers (see Fig. 6.1a).
b) I-PIPE primers (see Fig. 6.1b).
c) M-PIPE primers (see Fig. 6.1c).
3. PfwTurbo® DNA polymerase: 2.5U/U.1 (Stratagene, La Jolla, CA) (j«Note2).
4. Cloned Pfu DNA Polymerase Reaction Buffer (lOx): 200mM Tris-HCl (pH 8.8), 20mM MgS04, lOOmM KCl, lOOmM (NH4)2S04, 1% Triton® X-100, 1 mg/ml nuclease-free BSA (Stratagene, La Jolla, CA).
5. lOmM dNTP mix (contains all four dNTPs at lOmM each).
6. Milli-Q water.
7. PIPE Pfu Master Mix (lx): 5ul of lOx Cloned Pfu DNA Polymerase Reaction Buffer, 2.5 units of PfwTurbo® DNA polymerase, 1 ul of 10 mM dNTPs to a volume of 35 ul. (Template DNA (1-5 ng) and forward and reverse primers are added separately and the reaction is brought up to a final volume of 50 ul.).
8. Thermocycler: MJ Research PTC-200 (Bio-Rad Laboratories, Hercules, CA) or similar.
6.2.4. Transforming Competent Cells and Plating Transformed Cells
1. Competent cells (from Section 6.2.1).
2. Water bath (42°C).
3. 96-Well microtiter plates: Falcon® U-bottom, polystyrene plates (Becton Dickinson, Franklin Lakes, NJ).
4. Selective LB agar plates (from Section 6.2.2).
5. Sterile glass beads, 5-mm diameter, autoclaved.
6. Two to five sterile glass beads are dispensed into each of the 48 wells of the selective LB agar BioAssay tray for spreading cultures.
6.2.5. Screening 1. LB Broth (see step 2 - Section 6.2.1).
Colonies by Diagnostic 2. 96-Well microtiter plates: Costar® flat-bottom, polystyrene PCR(dPCR) plates
3. 96-Well PCR plates: Costar® Thermowell®, polypropylene plates.
4. Thermocycler: MJ Research PTC-200 (Bio-Rad Laboratories, Hercules, CA) or similar.
5. Taq Reaction Buffer (lOx): lOOmM Tris-HCl (pH 8.8), 500 mM KCl, 15mM MgCL,, 0.01% (w/v) gelatin.
6. Taq DNA Polymerase (see Note 3).
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6.2.6. SYBR-PCR Assay (for Optional Use with the Insert-Specific Reverse Primer Amplification)
7. dNTPs (lOmM) (seestep 5 - Section 6.2.3).
8. Forward sequencing primer (50uM).
9. Reverse sequencing primer or an insert-specific reverse primer (50 uM) (see Note 4).
10. Cells cultured from isolated colony picks.
11. Milli-Q water.
12. Taq Master Mix (for 1 reaction): 5(0,1 lOx Taq Reaction Buffer, lul lOmM dNTPs, lul Taq DNA Polymerase, 0.5 ul pBAD forward primer, 0.5 ul pBAD reverse or universal insert-specific reverse primer and 39 jliI Milli-Q water.
13. Taq Master Mix (for 96 reactions): 510 jLtl lOx Taq Reaction Buffer, 102 ul lOmM dNTPs, 102ul Taq DNA Polymerase, 51 ul pBAD forward primer, 51 ul pBAD reverse (for dPCR) or a universal insert-specific reverse primer (for SYBR-PCR) and 3,978 ul Milli-Q water (see Note 5).
1. 96-Well microtiter plates: Costar® flat-bottom, polystyrene plates.
2. SYBRPCR products.
3. Quench Buffer: 10mM EDTA.
4. Dilution Buffer: 50 mM Tris-HCl (pH 8.0).
5. SYBR Buffer: 40 ul of 10,000x SYBR Green I Dye (Sigma) is diluted in 20 ml of Dilution Buffer to make a 20x solution (see Note 6).
6. Microtiter Plate Fluorescence Reader: Spectramax Gemini XS with SoftMax Pro 4.6 software or similar.
6.2.7. Preparation of dPCR Products for Sequencing
6.2.8. Glycerol Stock Archival of Putative Clones
1. PCR product (see Section 6.2.5).
2. Exo/SAP Solution (14): 0.5 ul Exonuclease I, 0.5 ul Shrimp Alkaline Phosphatase, 4.0 ul Milli-Q water (enzymes available through USB, Cleveland, OH) (seeNote 7).
3. Thermocycler: MJ Research PTC-200 (Bio-Rad Laboratories, Hercules, CA) or similar.
1. 96-Well microtiter plates: Costar* flat-bottom, polystyrene plates.
2. Glycerol (80% (v/v)): 1 kg of 100% glycerol (p= 1.25g/cm3) is diluted to 1 L with Milli-Q water, then autoclave.
3. Cultures (see step 5 - Section 6.3.5).
4. Aluminum foil lids: Biomek® Seal and Sample (#538619, Beckman Coulter, Fullcrton, CA) or similar.
5. Freezer (-80°C).
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6.3. Methods
The PIPE method is based on the observation that, contrary to popular assumption, normal PCR amplifications result in mixtures of products which are not fully double stranded (15). The 5' ends of such products are left variably unpaired by incomplete 5' —» 3' primer extension caused by sequence-specific stalling and changes in the reaction equilibrium (less dNTPs available, more template copies to synthesize) in the final cycles of PCR. These unpaired 5' ends on the PCR products are the same 5' ends on the synthetic amplification primers. Therefore, a simple oligonucleotide design rule can control the sequences of these ends in a way that promotes easy cloning and mutagenesis.
The first 15 bases on the 5' ends of the primers are designed to be directionally complementary such that the resultant PCR fragment(s) can anneal as desired and become viable plasmids upon transformation. In basic PIPE cloning, the vector is linearized by V-PIPE PCR amplification and contains two distinct 5' ends (Fig. 6.1a). The inserts are I-PIPE PCR amplified with primers which contain 5'sequence complementary to the two distinct ends of the amplified vector (Fig. 6.1b). In this manner, annealing occurs directionally and creates a viable plasmid. In basic PIPE mutagenesis (M-PIPE), the entire plasmid is amplified (Fig. 6.1c). The two primers used are designed to create the mutation (a substitution, deletion, or insertion) and to be complementary to each other so that the linearized PCR product can self-anneal to recreate a viable, mutant plasmid. The following protocol describes PIPE cloning and mutagenesis in a 96-well plate.
6.3.1. Preparation of 1 Competent Cells
2
3
4
5
6
. Start a 10-ml overnight culture of cells in LB Broth at 37°C for a 1-L batch.
. On the following morning, seed 1 L of LB Broth with the 10-ml overnight culture.
. Incubate the culture at 37°C while shaking at 250 rpm until the optical density measured at 600 nm (OD600) reaches 0.4-0.6. (The remainder of this preparation is done on ice or in the cold room.).
. Split the 1-L culture into 2 x 500 ml sterile, disposable Corning Erlenmeyer polycarbonate flasks and pellet the cells by chilled centrifugation at 2,500 x^for 20min (see Note 8).
. Decant and discard the LB Broth.
. Add MgCl2 Solution at one-tenth the original volume (100 ml) to the pellet.
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7.
8.
9.
10
11
12
13
14
15
6.3.2. Preparation 1 of Selective LB Agar Plates
2.
3.
4.
6.3.3. PCRs for 1. Amplifying Cloning Vectors and Inserts 2 or Generating Site-Directed Mutants
Resuspend the pellet by gentle swirling the MgCl2 Solution over the pellet. Pipette up and down to break up dislodged pellet, if necessary (see Note 8).
Incubate the resuspended cells on ice for 30min.
Pellet the cells by chilled centrifugation at 2,500 x g for 20min.
Decant and discard the supernatant.
Add CaCl2 + Glycerol Solution at one-fiftieth the original volume (20ml) to the cell pellet.
Resuspend the pellet by gentle swirling the CaCl2 + Glycerol Solution over the pellet. Pipette up and down to break up dislodged pellet, if necessary.
Divide the competent cells into 2 ml aliquots using 2-ml microcentrifuge tubes.
Flash freeze the aliquots in liquid nitrogen.
Store aliquots at -80°C.
LB Agar plates: 200 ml of liquefied LB Agar is supplemented with an appropriate antibiotic at 55°C and then poured into the BioAssay tray with the 48-well divider removed from the tray (see Note 9).
After the LB agar has been poured, the 48-well divider is placed back into the tray and partially submerged into the LB agar creating 48 separate squares.
The LB agar is allowed to solidify at room temperature.
The plates are then dried overnight by propping the lids slightly open to allow sufficient gas exchange while minimizing potential contamination.
Dilute the 50-n.M oligonucleotide primer stocks to 10 uM with Milli-Q water.
Set up the PIPE PCRs.
a) (V-PIPE) Tranfer 5ul of both 10uM forward and reverse primers (see step 2a - Section 6.2.3) into PGR tubes and then add 5ul of template (see step li - Section 6.2.3) and 35 ul of PIPE Pfu Master Mix (see Notes 10 and 11).
b) (I-PIPE) Tranfer 5ul of both lOuM forward and reverse primers (see step 2b - Section 6.2.3) into a PGR plate and then add 5 ul of template (see step lii - Section 6.2.3) and 35ul of PIPE Pfu Master Mix (^Note 10).
c) (M-PIPE) Tranfer 5 ul of both 10uM forward and reverse primers (see step 2c - Section 6.2.3) into a PGR plate and then add 5ul of template (see step liii - Section
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6.2.3) and 35 pi of PIPE Pfu Master Mix (see Notes 12 and 13).
3. Thermocycler conditions for PIPE amplifications.
a) (V-PIPE) 95°C for 2min, then 25 cycles of 95°C for 30s, 55°C for 45s and 68°C for 14min, and finally a 4°C hold.
b) {I-PIPE) 95°C for 2min, then 25 cycles of 95°C for 30s, 55°C for 45 s and 68°C for 3 min, and finally a 4°C hold (see Note 14).
c) (M-PIPE) 95°C for 2 min, then 25 cycles of 95°C for 30s, 55°C for 45 s and 68°C for 14min, and finally a 4°C hold.
4. Confirm successful amplifications by gel electrophoresis.
3. Transfer 2 pi of each of the PCRs into wells of a prechilled microtiter plate.
- When cloning, first mix 2 pi from the V-PIPE and 2 pi I-PIPE reactions together (see Note 15).
- For mutagenesis, 2 pi from the M-PIPE reaction can be used directly (see Note 16).
4. Dispense 20 pi of competent cells into each well. Pipette up and down ONCE to ensure DNA has mixed with the cells.
5. Incubate the DNA-cell mixture on ice for 15 min.
6. Heat shock the cells by floating the microtiter plate in a 42°C water bath for 45 s (see Note 17).
7. Immediately return the microtiter plate to ice.
8. Dispense 100 pi of LB Broth (no antibiotic) into each well.
9. Recover the transformed cells by incubating at 37°C while shaking at 250 rpm for lh.
10. Dispense 100 or 40 pi of the recovered cells into the respective wells of the selective LB agar trays with glass beads.
11. Shake (by hand) the trays enough to move the glass beads and evenly distribute the cells across the entire well.
12. Invert the tray to drop the glass beads off of the LB agar and onto the lid.
13. Remove the glass beads from the lid.
14. Incubate the inverted trays overnight (12—16h) in a stationary 37°C incubator to grow the bacterial colonies (see Note 18).
6.3.4. Transforming Competent Cells and Plating Transformed Cells
2
1
Thaw 2 ml aliquot of competent cells (see step 15 - Section 6.3.1) on ice for 10-15min.
Chill a 96-well microtiter plate on ice for 10-15 min.
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6.3.5. Screening Colonies by Diagnostic PCR(dPCR)or SYBR-PCR
1. Dispense 200 ul of LB Broth with appropriate antibiotic into the wells of flat-bottom 96-well plate.
2. Using aseptic technique, pick and transfer 1-4 isolated colonies per transformation (see step 14 - Section 6.3.4) into unique wells of the microtiter plate.
3. Incubate the plate at 37°C while shaking at 250rpm for at least 3h (up to overnight).
4. Transfer 3-ul samples from each culture into a 96-well PCR plate.
5. Put the remainder of the cultures (~197ul/well) back into the shaking incubator to continue growth for future glycerol stock archival.
6. Add 47 ul of Taq Master Mix to each well containing cells in the PCR plate (wNote 19).
7. Place the PCR plate into a thermocycler.
8. Amplify the DNA fragments using the following cycling conditions: 95°C for 2min followed by 30 cycles of 95°C for 30s, 55°C for 45s and 72°C for 3min, then finally a 4°C incubation.
6.3.6. SYBR Assay (for Optional Use with the Universal Insert-Specific Reverse Primer Amplification)
6.3.7. Preparation of dPCR Products for Sequencing
6.3.8. Glycerol Stock Archival of Putative Clones
1. Dispense 50ul of Quench Buffer in a flat-bottom 96-well plate.
2. Transfer 5 ul of each SYBR-PCR product (see step 8 - Section 6.3.5) into the wells with Quench Buffer (see Note 20).
3. Dispense 150ul of SYBR Buffer into each well.
4. Prepare an unamplified sample for a negative control.
5. Measure fluorescence of each sample using a microtiter plate fluorescence reader. Excitation: 485 nm. Emission: 525 nm. Auto-cutoff enabled.
6. SYBR results are determined on a relative scale with the positive wells having at least fourfold higher fluorescence than the negatives or the control.
1. Dispense 5ul of Exo/SAP Solution15 directly into the dPCR positive wells (determined by gel analysis, SYBR assay, or simply assumed to be positive).
2. Incubate the Exo/SAP reaction at 37°C for 30min, then 75°Cfor 15 mill.
3. Submit these samples directly for sequencing.
1. Add 50 |il of 80% (v/v) glycerol (20% final) to 150ul of each culture (see step 5 - Section 6.3.5) in a 96-well microtiter plate.
2. Mix the glycerol into the culture by pipetting up and down.
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3. Seal the plate using the aluminum foil lid.
4. Store the plate in a -80°C freezer.
6.4. Notes
1. Milli-Q water is purified to a resistivity of 18.2 MQ cm and contains total organics at less than five parts per billion using the Milli-Q Synthesis System (Millipore, Billerica, MA).
2. Thermostable DNA polymerases from Pyrococcus furiosus {Pfu), Thermococcus kodakaraensis (KOD), and Thermus aquaticus(Taq) as well as Phusion" DNA Polymerase have all been used successfully. However, the majority of our experience and, therefore, success has come from using P/wTurbo DNA Polymerase. We also observe spurious mutations using Taq DNA Polymerase which have not been observed using the proofreading enzymes.
3. Taq DNA Polymerase works very well for amplifying DNA directly from cell cultures. The ability to PGR from these cells grown from isolated colonies eliminates the need to miniprep DNA for sequencing. Amplifying DNA from cell cultures using P/wTurbo DNA Polymerase has NOT worked well for us.
4. The insert-specific reverse primer is designed such that successful PCR amplification is dependent on the insert annealing to the vector to form a viable colony. Conditional PCR can be used in series with a fluorescence assay (SYBR) to determine insert-containing plasmids without running a DNA gel.
5. This master mix is actually made at 102x to account for volume losses in the multiple transfer steps.
6. Although SYBR dyes are reported to be far less carcinogenic than ethidium bromide, they still bind tightly to DNA. Appropriate care should be observed in handling and disposal.
7. Alternate Exo/SAP protocols may use 4 ul of buffer instead of 4ul of water, but our sequencing results have not suffered.
8. Use centrifugation vessels which maximize the surface area across which the cells are pelleted. This makes it much easier to resuspend the cells without excessive stress to the cells.
9. Subjecting antibiotic(s) to the LB agar at temperatures greater than 55°C for extended periods of time can decrease relative effectiveness on susceptible cells.
10. Our vector pSpeedET contains the ccdB gene which is toxic to our expression strain (HK100). This creates a negative
The Polymerase Incomplete Primer Extension 101
selection against vector template DNA. Strains carrying the ccdA antidote on the F' episome are not susceptible to this negative selection. In the V-PIPE PGR, the entire plasmid is amplified except for the ccdB cassette.
11. For large cloning projects, amplifying the vector separately from the inserts is logistically best. It is possible, however, to amplify both the vector and the insert (from the same or different templates) in the same reaction (IV-PIPE). This is done by adding both templates and all four primers to the same 50 pi PCR. Each primer is added at half the original concentration so that the total primer concentration in the reaction remains fixed at 2pM.
12. Mutagenesis is not limited to a single substitution, deletion, or insertion per reaction. We have made up to five substitutions using a single primer pair when the sites are all in close proximity. We have made four codon substitutions using four primer pairs in the same reaction when the sites were disbursed. We have made N- and C-terminal insertions and deletions separately and simultaneously. In all cases, the total primer concentration in the reactions is 2pM.
13. In most cases, the M-PIPE templates encode the same antibiotic resistance as the desired mutant plasmids will have. We have found that ~lng (20pg/reaction) is typically low enough to eliminate background transformants. In the cases where PCR amplification is only successful using higher amounts of template, Dpnl may be used to digest the template DNA to reduce background.
14. Use the thermocycling conditions for V-PIPE PCR when trying to amplify the vector and insert in the same reaction.
15. The PCR products are used directly out of the thermocycler. There are no post-PCR treatments to the PCR products unless the background is determined to be too high (Dpnl treatment, Note 13). If the vector and the insert were amplified together (IV-PIPE), then 2 pi from that reaction is sufficient.
16. The PCR products are used directly out of the thermocycler. There are no post-PCR treatments to the PCR products unless the background is determined to be too high (Dpnl treatment).
17. Efficient heat shock requires direct water to well contact. Be careful to avoid introducing air pockets between the plate and water.
18. If satellite colonies are present, incubate the trays overnight at 30°C instead of37°C.
19. For SYBR-PCR the universal insert-specific reverse primer is designed to anneal to any amplified DNA insert. PCR amplification is conditional on the insert fragment annealing to the
102      Klock and Lesley
vector in the correct orientation. Therefore, PGR is successful on insert-containing plasmids but not with background trans-formants such as vector only or PGR template contamination.
20. This assay is not helpful when using two vector-specific primers since amplification will occur with or without the insert. Although the assay is very quick in identifying insert-containing plasmids (compared to agarose gel), the sizes of the PCR products cannot be determined. In our experience, the SYBR assay and PCR products of the correct size have at least an 80% correlation.
Acknowledgments
This work was supported, in part, by NIH grant PSI U54 GM074898. We thank Eric Koesema for his experiments in confirming the proof of principle and testing the initial applications of the PIPE method. We also thank Mark Knuth, Christian Ostermeier, and Roger Benoit for helpful discussions.
References
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3. Aslanidis, C, de long, P.J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069-74.
4. Hsiao, K. (1993) Exonuclease III induced ligase-free directional subcloning of PCR products. Nucleic Acids Res. 21, 5528-9.
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construction of recombinant DNA without restriction enzymes. Curr Biol. 8, 1300-9.
8. Oliner, J.D., Kinzler, K.W., Vogelstein, B. (1993) In vivo cloning of PCR products in E. coli. Nucleic Acids Res. 21, 5192-7.
9. Hardey, J.L., Temple, G.F., Brasch, M.A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788-95.
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12. Kirsch, R.D., Joly, E. (1998) An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res. 26, 1848-50.
13. Sawano, A., Miyawaki, A. (2000) Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28, e78.
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Hanke, M., Wink, M. (1995) Direct DNA sequencing of PCR- amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques. 18,636.
15. Olsen, D.B., Eckstein, F. (1989) Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing. Nucleic Acids Res. 23,9613-20.