Team:TU Darmstadt/Notebook/sec1/K1602059
K1602059 - B0034-Xylanase (B0034-xynA)
The gene xynA encoding the enzyme Xylanase from Bacillus subtilis was amplified with the oligonucleotides xynA FW and xynA REV. To delete an internal cutting site of PstI a Quickchange PCR® was performed using the oligonucleotides xynAmut FW and xynAmut REV. After confirmation of the PCR product by gel electrophoresis (Figure 1) and DpnI digest, the construct was digested with EcoRI and PstI and ligated into pSB1C3. E. coli Top 10 were transformed via heat shock. Afterwards, colonies were screened using a colony PCR (Figure 2). Positive colonies were inoculated and the plasmid was extracted. The construct was digested using XbaI and PstI and ligated into B0034-pSB1A2 followed by E. coli Top 10 transformation. The screening by colony PCR showed positive colonies (Figure 3).
Figure 1 Standard PCR of xynA (1 + 2). The size of the amplified product was around 0.7 kbp. DNA marker: 2-Log DNA Ladder (NEB).
Figure 2 Quickchange PCR of xynA (2). The size of the amplified product was around 3.0 kbp. DNA marker: 2-Log DNA Ladder (NEB).
Figure 3 Colony PCR of xynA cloned into pSB1C3 (1 + 2). The size of the amplified product was around 0.7 kbp. DNA marker: 2-Log DNA Ladder (NEB).