Isolation of Plasmid DNA Through Mini-Prep
Objectives
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Brainstorm reasons why researchers or pharmaceutical companies would want
to isolate plasmid DNA.
Isolate and purify a cloned plasmid by performing a mini-prep procedure.
Classroom Time Requirement
Additional time for teacher preparation will be needed; see Pre-Lab Teacher Preparation.
Day 1: Perform mini-prep experiment - 45 minutes
Materials Included in Kit
This kit includes materials for 30 students working in pairs. Pairs of students share
materials, but each student conducts his or her own experiment.
 30 tubes*
Luria broth, containing 500l each
 1 vials
Ampicillin, 20 mg
 2 ml
Sterile distilled water (for ampicillin)
 15 tubes**
Cell Resuspension Solution, containing 440 l each
 15 tubes**
Cell Lysis Solution, containing 440 l each
 15 tubes**
Neutralization Solution, containing 440 l each
 15 tubes**
DNA Purification Resin Solution, containing 2.2 ml each
 15 tubes**
Column Wash Solution, containing 3 ml each
 1 bottle
95% Ethanol
 15 tubes**
TE Buffer, containing 120 l each
 30
Luer-Lok columns
 30
Syringes, 3mL
 90
Microcentrifuge tubes
 1 pack
Sterile Loops
* one for each student
** one for each pair of students
Materials Needed But Not Included in Kit
For Prep and Storage
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 37 C incubator
For Lab
 Agar plate with transformed E. coli bacteria (These transformed bacteria come from the
Transformation Lab)
 Microcentrifuge
 Markers
 Microcentrifuge test tube rack
 Foam rack
 p20, p200 micropipettes
Copyright  2007 MassBioEd
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Micropipette tips
10 mL graduated cylinder
15 tubes TE Buffer, containing 120 l each
Pre-Lab Teacher Preparation
Prepare Cultures
 Add 2 mL sterile distilled water to the vial of ampicillin.
 Cap and shake gently to mix.
 Using sterile technique, add 50 l of the ampicillin solution to each of the tubes of
Luria broth provided. Label these tubes “LB/Amp Broth”
 Use sterile loops to obtain transformed E. coli cells containing a plasmid from a
Petri plate to the LB/Amp Broth tubes you made. (E. coli bacteria are not
supplied with the kit. They come from the Transformation Lab)
 Incubate E. coli in LB/Amp tubes for 16-24 hours at 37°C.
Dilute Column Wash Solution with Ethanol
 Add 1.5 mL of 95 % Ethanol to 3 mL of column wash buffer
Lab Station Set-Up
Materials Needed Per Pair
 Microcentrifuge rack containing
o 2 liquid cultures of E. coli in 550 l of LB/Amp Broth
o 6 empty, sterile microcentrifuge tubes
 Marker
 Microcentrifuge
 Liquid waste containers
 p20 and p200 micropipettes
 Pipet tips
 1 tube
Cell Resuspension Solution, containing 440 l
 1 tube
Cell Lysis Solution, containing 440 l
 1 tube
Neutralization Solution, containing 440 l
 2
Miniprep columns
 2
Syringes, 3mL
 1
10 mL graduated cylinder
 1 tube
DNA Purification Resin Solution, containing 2.2 ml
 1 tube
Column Wash Solution+ Ethanol, containing 4.5 ml
 1 tube
TE Buffer, containing 120 l
Copyright  2007 MassBioEd
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