2 Mini-Prep:A rapid, small scale method of obtaining or retrieving plasmid DNA (plasmid DNA + foreign/inserted DNA) from bacterial cells.2 General Variations:1. Boiling Method2. Alkaline Lysis Method (With a modification to collect plasmids)
3 Overall Goals: To lyse cells using Alkaline Lysis Method To precipitate out proteins and bacterial DNA using Sodium AcetateTo separate plasmid from solution with a column. To freeze plasmid DNA for further study
4 Preparation. Grow the bacteria Grow an overnight (ON) culture of the desired bacteria in 2-5 ml of LB medium containing the appropriate antibiotic for plasmid selection. Incubate the cultures at 37°C with vigorous shaking.p. 1-12
5 2a. Transfer the cells to a tube and centrifuge Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (12,000 rpm) in the microcentrifuge.First tap or gently vortex the glass culture tube to resuspend the cells which have settled. The culture can be transferred to the microfuge tube by pouring.(Follow steps in Lab 6)p. 1-12
6 2b. Remove the supernatant Remove the growth medium (supernatant or sup) by aspiration or by using the P-1000.Leave the bacterial pellet as dry as possible so that additional solutions are not diluted.p. 1-12
7 Important Solutions and Their Purpose: Solution 1: GTE (Glucose Tris EDTA):Tris is a buffer that works at physiological pH (pH = 7.4).EDTA binds divalent cations in the lipid bilayer, thus weaking the cell membrane and also protecting DNA from DNAses by removing their cofactors.Solution 2: SDS/NaOH (Sodium dodyclsulfate + Sodium Hydroxide) - Alkaline Lysis Solution:The detergent SDS(Soap) dissolves the lipid component of the cell membrane (plasma membrane), as well as cellular proteinsThe NaOH denatures the chromosomal and plasmid DNA into single strands (ssDNA). The intact circle of plasmid DNA remain interwined.
8 Important Solutions and Their Purpose: Solution 3: KOAc (Potassium Acetate + Acetic Acid):The Acetic Acid returns the pH to neutral, allowing DNA strands to renature. The large, disrupted chromosomal DNA strands can not rehybridize perfectly, but instead collapse into a partially hybridized tangle.At the same time, the Sodium Acetate precipitates the SDS from the cell suspension along with proteins and lipids with which it has associated. The renatured chromosomal DNA is trapped in the SDS/Lipid/Protein precipitate. Only smaller plasmids DNA and RNA molecules escape the precipitate and remain in solution.
10 Important Solutions and Their Purpose: Wash solution-note the smell of alcohol (it is 80% Ethanol)-Washes through contaminants (ie. small RNAs while the plasmid DNA stays bound to the column's silica filter.FYI: if doing “old fashion” mini prep w/ no column, you must add Rnases to the protocolSpinDiscard“flow through”that containscontaminants
11 Important Solutions and Their Purpose: Drying step-Is essential to remove as much of the Ethanol as possibleIf skipped many complications for use of plasmid later. For example:-can not run plasmid on gel for basic analysis-can not sequence Plasmid DNA-DO NOT SKIP!!!SpinDiscard“flow through”that containscontaminants
12 Important Solutions and Their Purpose: Elution Solution. This low salt buffer will cause the plasmid to release from the column and come off in the spin.Switch outcollection tube for1.5ml flip top MCT
13 Step by Step 1. Spin the culture and remove LB broth 2. Resuspend in GTE3. Lyse in Soap and Base (SDS/NaOH)4. Neutralize to renature the plasmids5. Spin to remove Genomic DNA and cell debris.6. Transfer Supernatant to column and spin7. Wash by spining with Wash buffer8. Elute and Collect Plasmids