Haynes Lab:Notebook/Engineering PC-TFs/2012/11/29

Summary

• Repeformed miniprep on psb1a2 vector.
• Add 10 μl of 7X lysis buffer to 600 μl of E. coli cμlture.
• Add 350 μl of cold Neutralization Buffer
• Centrifuge at 11,000-16,000 x g for two minutes.
• Transfer the supernant in the Zymo-Spin column.
• Place the column in to a Collection tube and collection tube and centrifuge for 15 seconds. *Discard the flow-through and place the column back into the same Collection tube.
• Add the 200 μl of Endo Wash Buffer to the column. Centrifuge for 15 seconds.
• Add 400 μl of Zyppy Wash Buffer to the column. Centrifuge for 30 seconds.
• Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 μl of Zyppy Elution Buffer directly to the column matrix and let stand for one min at room temperature Centrifuge for 15 seconds to elute the DNA.

Plan for tomorrow: Perform plasmid digest on psb1a2 vector. If digest does not succeed, will streak plasmid bacteria on plate. I will ask Vi for digest DNA.