Procedure
This kit uses two step PCR to generate template DNA. The procedure is described below.
1) 1st PCR: The DNA encoding a protein of interest will be amplified. The PCR product will contain start and stop codon, whole coding region of the protein, and additional sequences for the 2nd PCR.
The primers for this step are gene specific primers with overhangs and can be ordered on the Bioneer's website.
2) 2nd PCR: Using a product from the 1st PCR, 2nd PCR performed using primer set by which T7 promoter and RBS are introduced at its 5’ end and T7 terminator is added at its 3' end.
PCR product after 2nd PCR should be gel-purified for the cell-free protein synthesis.
Experimental results
Figure 1. Agarose gel data of template DNA synthesized by each PCR
Synthesis of linear template DNA with ExiProgen™ ProXpress PCR Template Kit.
M1; 100bp DNA Ladder(Bioneer, D-1030), M2; 1kb DNA Ladder(Bioneer, D-1040)
1; SAV (Template – pT), 2; RNase H (Template – BL21(DE3) gDNA), 3; hGH (Template – pT)
4; CAT (Template - pBIVT), 5; UDG (Template - BL21(DE3) gDNA), 6; AcGFP (Template – pBIVT),
7; EVO (Template – pT), 8; RFP (Template – pIVEX), 9; Poly A polymerase (Template – pET15b),
Figure 2. PAGE gel data for synthesized proteins.
Each linear template DNA generated by ExiProgen™ ProXpress PCR Template Kit are used as template for protein synthesis with ExiProgen™ EC Protein Synthesis Kit.
M; AccuLadder™ Protein Size Marker (Low)(Bioneer, D-2020),
1; SAV (13 kDa), 2; RNase H (20 kDa),3; hGH (23 kDa), 4; CAT (26.5 kDa), 5; UDG (28 kDa), 6; AcGFP (28 kDa),7; EVO (30 kDa), 8; RFP (31kDa), 9; Poly A polymerase (54 kDa)
Kit contents
| AccuPower® ProFi Taq PCR Premix (20ul) |
96 tubes |
192 tubes |
| N terminus upstream cassette (5 ng/ul) |
5 ul x 16 tubes (Yellow, NU) |
5 ul x 32 tubes (Yellow, NU) |
| N terminus downstream cassette (5 ng/ul) |
5 ul x 16 tubes (Yellow, ND) |
5 ul x 32 tubes (Yellow, ND) |
| C terminus upstream cassette (5 ng/ul) |
5 ul x 16 tubes (Violet, CU) |
5 ul x 32 tubes (Violet, CU) |
| C terminus downstream cassette (5 ng/ul) |
5 ul x 16 tubes (Violet, CD) |
5 ul x 32 tubes (Violet, CD) |
| 2nd Forward primer (10 pmoles/ul) |
5 ul x 16 tubes (White, 2F) |
5 ul x 32 tubes (White, 2F) |
| 2nd Reverse primer (10 pmoles/ul) |
5 ul x 16 tubes (White, 2R) |
5 ul x 32 tubes (White, 2R) |
Storage condition
at - 20°C