What is the purpose of plasmid miniprep?
Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes, but should not be used with out additional cleanup for embryonic injections.
Why is it called miniprep?
Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method. The extracted plasmid DNA resulting from performing a miniprep is itself often called a “miniprep”.
How do you make a plasmid miniprep?
- Take 0.5ml of overnight E. coli culture in a microfuge tube.
- Add 0.5ml of phenol:chloroform:isoamylalcohol (25:24:1).
- Mix by vortexing at the maximum speed for 1 minute.
- Spin at 12,000g for 5 minutes.
- Pour off the supernatant, add carefully 0.5ml of 70% ethanol to the side of the tube, pour off.
Why should you not vortex when performing a plasmid miniprep?
Close the caps and mix the solutions by rapidly inverting them a few times. DO NOT VORTEX since the chromosomal DNA released from the broken cells could be sheared into small fragments and contaminate your plasmid prep. SDS is an acronym for Sodium Dodecyl Sulfate.
How can you separate a plasmid from another DNA?
An alkaline solution containing sodium dodecyl sulfate (SDS) is then added to facilitate cell lysis and the complete denaturation of both genomic and plasmid DNA along with all the proteins in the solution. A potassium acetate solution is then used to neutralize the sample and separate the plasmid DNA from the gDNA.
How do you isolate plasmid DNA from E coli?
- Inoculation. Inoculate 5 mL LB, supplemented with appropriate antibiotics, using a single colony. Grow over night at 37˚C.
- Cell harvest. Centrifuge culture at maximum speed and RT for 1 min.
- Cell lysis. Completely remove supernatant.
- Isopropanol precipitation. Add 1 volume of isopropanol.
- DNA recovery.