Hi, Im trying to obtain the alpha-amylase gene from E. coli 25922 & E. coli 8379 and ligate it with pGLO plasmid which would then be transformed into E. coli HB101 cells.

Could anyone correct my workflow if its wrong?

1. Perform DNA Extraction on E. coli 25922 & E. coli 8379

2. Gel electrophoresis to confirm successful DNA extraction

3. PCR to amplify alpha amylase gene

4. Gel electrophoresis to confirm amplification of amylase gene

5. Once confirmed carry out PCR cleanup

6. Restriction digestion performed on both amylase & pGLO plasmid

7. Perform gel electrophoresis to ensure restriction digestion is successful

8. Ligation of alpha amylase with pGLO plasmid

9. Transformation

10. Protein extraction from the cells

11. SDS page

14. Gel extraction to obtain amylase product

15. Western blot to confirm presence of amylase

I have also designed my PCR Primers for E. coli 25922 & 8379, I would really appreciate it if anyone could take a look at them to see if improvements can be made.