Tag: research

Molecular work 01: Plasmid extraction:

For plasmids, gDNAs, RNAs etc extraction, nowadays, we use commercial kits. I do not know when we started using commercial kits, but in early 2000s, many laboratories still used non-commercial methods- lab made Sol I (lysozyme lysis buffer), II (Acid buffer) and III (Alkaline buffer), following Phenol, Chloroform, and Isopropanol clearance after extraction. The most successful companies for plasmid kits may be Quiagen and Macherey-Nagel. They have plasmid kids for Mini, Midi, Maxi, Giga, Endo toxin free! In my mind, MN yields were better than Quiagen, but Giga prep I have used only Qiagen kit, so I am not sure whether MN has Giga prep kits as well. Quiagen, Promega and MN are still the most successful companies I think. Quiagen’s market occupancy started before 2000 even in South Korea. Also someone who is old like me might know and used CsCl plasmid purification. What I learned first during my Master degree training, was plasmid purification using CsCl ultracentrifuge method. It was simple but took time. My Master degree mentor Dr. Seho Park preferred CsCl at that time. I do not know which method he likes currently.

A few years ago, I saw a biotech company where they work to make a plasmid isolation machine. I’m not quite sure whether they were successful. However, you may hear Thermo KingFisher Plasmid Pro that I never used on my hands. Also you may hear automated lab components provided from Molecular device. I am not sure how they are successful. Long time ago, we/some old generation like me still remember that even we manually moved PCR tubes from different temperature water baths to another for PCR reaction.

About the water

Last step is adding solution either TE (Tris EDTA) or water. I recommend that you have to keep in mind what you are doing, for the next steps, before adding either TE or water. Generally I recommend adding water for the next steps: Restriction enzyme treatment, in vitro (cell), and in vivo (animal) treatment etc. TE definitely is a good solution for storage, but if you keep your plasmid DNAs at -20 °C, they are really safe, not degrading. You also could aliquot them. Usually, if you use water, you could save/reduce amounts of restriction enzyme using small reaction volume. Endo toxin free molecular grade water is easy to purchase from Fisher.

GMP plasmids

Maybe some heard about GMP (Good manufacturing practice) plasmids. I just googled how many GMP plasmids supplying companies are globally. There are quite numbers of companies including Aldevron and Catalent.