(Practice)Extraction of plasmid using the Alkaline extraction method (shown in fig.1).

fig.1 pTAC2 including insert

Extraction of Plasmid, pTAC2 including insert, using the Alkaline extraction method, RNase(100μg/mL) was used. RNA fragments were seen at the bottom of the gel, furthermore, desired band was observed (shown in fig.2).

fig.2 pTAC2 including insert

Extraction of plasmid, pTAC2 including insert, using the PEG precipitation. RNA was not seen while desired band was clearly visible (shown in fig.3).

fig.3 pTAC2 including insert

Extraction of plasmid, pTAC2-M. ApeKI, using the PEG precipitation. RNA was not seen while desired band was clearly visible (shown in fig.4).

fig.4 pTAC2 including insert

Extraction of plasmid, pMD20 from E. coli　JM109, using the PEG precipitation. RNA was not seen while desired band was clearly visible (shown in fig.5).

fig.5 pMD20 from E. coli

Previously extracted plasmid, pMD20, digested by the restriction enzyme, SmaI. Calculations of molecular weight done via ImageJ (shown in fig.6 and table 1)

fig.6 pMD20 after digestion by SmaI

Gel extraction of digested plasmid, pMD20.

Table 1 pMD20 (SmaI+gel purification)

Extraction of plasmid, pTAC2 in JM109, using the PEG precipitation. RNA was not seen while desired band was clearly visible (shown in fig.7).

fig.7 pTAC2 from JM109

Desired band of HMG-CoA synthase is visible (shown in fig.8)

fig.8 HMG-CoA Syntase

left side - Shows the bands of pMD20 reductase front plasmid. The ride side – Shows the bands of pTAC digested by EcoRI, and PstI. The result is shown in fig.9. Bands of Synthase after being digested by EcoRI and PstI

fig.9 pMD20 reductase front plasmid, Synthetase and pTAC after digestion by EcoRI and Pst1

Gel extraction of digested plasmid, pTAC, and synthase (shown in fig.10).

fig.10 Gel extraction of digested pTAC and Synthase

Left side-pTAC and Synthase Right side-PCR product of Reductase front in pMD20 (To check whether recombination was successful) and iPCR to amplify pMD20 including a part of reductase to introduce the remaining sequence of reductase (shown in fig.11).(fail)

fig.11 pTAC and Synthase. PCR and iPCR of reductase front in pMD20.

iPCR to cut pMD20 to introduce the remaining sequence of reductase (shown in fig.12). (fail)

fig.12 iPCR of reductase front in pMD20

PCR of Reductase front in pMD20(To check whether recombination was successful )(shown in fig.13).

fig.13 PCR of reductase front in pMD20