Control experiments were conducted using mouse (ICR) cDNA, genomic DNA or bacteria genomic DNA. Three target specific primers (TSP1, 2 and 3) for each gene were designed to amplify unknown target sequences adjacent to the known sequences. The first PCR reaction was performed using DW-ACPs and TSP1 primer. In the second and third PCR reactions, DW-ACP-N and TSP2, and Universal primer and TSP3 were used as described in sections 5B and 5C, respectively.

Fig. 3. Various applications of DW ACP-PCR™.
A. PCR amplification products for mouse TNF-a promoter region using DNA Walking SpeedUp™ Kit.
B. The amplification of the 5'-end region sequences of bacterial argC promoter region using DNA Walking SpeedUp™ Kit.
C. The amplification of the 5'-end region sequences of mouse PBP cDNA (EST) using DNA Walking SpeedUp™ Premix Kit. Each of four different DW-ACP (lane 1), DW-ACP2 (lane 2), DW-ACP3 (lane 3), and DW-ACP4 (lane 4) generated one major product showing a different size. These products were identified as the 5-end region sequences of PBP cDNA by sequence analysis. M: Forever 100 bp Ladder apersonaliser.

* Control PCR (mouse TNF-a)

1st PCR

Components Vol. (µl) Conditions   Template (50ng/µl DNA) 2 10 X buffer (+15mM MgCl2) 5 dNTP (2mM) 5 Primer (DW-ACPs) 2.5µM 4 Primer (TSP1) 10µM 1 Taq (Roche Taq) 5u/µl 0.5 DW 32.5 Final 50ul

* uM=pmole/µl

2nd PCR

Components Vol. (µl) Conditions   Template (purified 1st PCR) 2 10 X buffer (+15mM MgCl2) 5 dNTP (2mM) 5 Primer (DW-ACPN) 10µM 1 Primer (TSP2) 10µM 1 Taq (Roche Taq) 5u/µl 0.5 DW 35.5 Final 50ul

* uM=pmole/µl

3rd PCR

Components Vol. (µl) Conditions   Template (2nd PCR) 1 10 X buffer (+15mM MgCl2) 5 dNTP (2mM) 5 Primer (U-primer) 10µM 1 Primer (TSP3) 10µM 1 Taq (Roche Taq) 5u/µl 0.5 DW 36.5 Final 50ul

* uM=pmole/µl