MINIPREP EXPRESSTM Protocol
Revision No. 2000-999-5D02W



Introduction

The Miniprep ExpressTM is the first truly cost-effective plasmid miniprep. At only pennies per prep, it is the frequent cloner's ideal tool for screening hundreds of recombinant clones in record time using restriction enzyme analysis and PCR.

The Miniprep ExpressTM, which consists of the Miniprep Express MatrixTM**, is designed to complement the alkaline lysis (1,2) and boiling minipreps (3) procedures. Simply add 400 µl of Miniprep Express MatrixTM to a crude plasmid prep; wash; air dry, and elute the DNA in 75 µl H2O or TE.

**The Express MatrixTM contains guanidine thiocyanate; use with proper precaution.



Lysing Methods
  1. Alkaline Lysis(1,2)
    1. Pellet 1 ml of overnight culture; remove and discard the supernate. Remove the medium by aspiration or pour it off and remove the remaining drop with a pipet tip. (To make an aspirator, attach a disposable 200 µl pipet tip to a vacuum line with a trap in between).
    2. Add 100 µl Solution I and mix by vortexing until the cells are completely resuspended.
    3. Add 200 µl Solution II and invert a few times to mix.
    4. Add 150 µl Solution III; vortex 1 second and microfuge 2 minutes. To prevent possible genomic DNA contamination when isolating low copy number vectors, vortex at a lower speed or shake moderately a couple of times until a uniform white precipitate is observed.
    5. Transfer supernate to a clean tube and continue with Miniprep ExpressTM protocol.
  2. Boiling Prep(3,4)
    1. Pellet 1 ml of overnight culture; remove and discard the supernate. See Alkaline Lysis, step a.
    2. Resuspend cells in 300 µl STET and add 25 µl of lysozyme.
    3. Incubate 2-5 minutes on ice and place 1-2 minutes in a boiling water bath.
    4. Microcentrifuge 5 minutes; transfer supernate to a new tube and continue with Miniprep ExpressTM protocol.



Miniprep ExpressTM Protocol

a. Add 400 µl Express Matrix (mix solution completely before each use) and invert ~5 times to mix. Plasmid DNA binds instantaneously to the matrix.

b. Microfuge 5-10 seconds at room temperature and decant supernate.

c. Add 500 µl of 80% ethanol and resuspend the Matrix/DNA complex by gently stirring a couple of times with a 200 µl pipet tip. Complete resuspension is not necessary for efficient washing.

d. Spin 5-10 seconds and decant wash.

e. Pulse spin; remove last drop of ethanol with a pipet tip and air dry for 5 minutes at room temperature. Longer drying periods, 5 minutes to overnight, have no effect on the DNA quality and its recovery from the matrix.

f. Resuspend the dried Matrix/DNA complex in 75 µl H2O or TE by gently stirring with a pipet tip until a uniform suspension is obtained. Complete resuspension ensures maximal DNA recovery. Spin 1 minute and transfer supernate containing plasmid DNA to a new tube. DNA is ready for restriction enzyme and PCR analysis.




Solutions

Solution I
50 mM Glucose
25 mM Tris.Cl pH 8
10 mM EDTA pH 8		 Solution II
0.2 M NaOH
1% SDS		 Solution III
60 ml, 5 M potassium acetate
11.5 ml glacial acetic acid
28.5 ml H2O
STET
0.1 M NaCl
10 mM Tris.Cl pH 8
1 mM EDTA pH 8
5% Triton X-100		 Lysozyme
10 mg/ml in 10 mM Tris pH 8



References

  1. Sambrook, J., et al., eds. (1989) Molecular Cloning- A Laboratory Manual. 2nd edition, Cold Spring Harbor Lab. Press, 1.25-1.28.

  2. Ausubel, F.M. et al., eds. (1991) Current Protocols in Molecular Biology. Wiley Interscience, New York, 1.6.1.

  3. Sambrook, J., et al., eds. (1989) Molecular Cloning- A Laboratory Manual. 2nd edition, Cold Spring Harbor Lab. Press, 1.29 - 1.30.

  4. Ausubel, F.M. et al., eds. (1991) Current Protocols in Molecular Biology. Wiley Interscience, New York, 1.6.4.



Utilizing Miniprep Expressª in Screening Strategies

The Miniprep ExpressTM is ideal for screening a large number of recombinant clones. (See pp 325-326 for subcloning strategies)

  1. Prepare master plate and grow overnight cultures.
    1. Place a master plate (LB/CircleGrow + antibiotic) on the grid (see next page) and draw an arrow on the side of the plate corresponding to the mark on the grid.
    2. Pick colonies from the transformation plates with sterile toothpicks or autoclaved pipet tips; inoculate the master plate (one colony per numbered sector); and place the toothpicks/pipet tips in numbered culture tubes containing liquid media + antibiotic.
    3. Shake cultures overnight at 37°C and process the next day. Incubate master plate inverted at 37°C and grow overnight until colonies are observed. Seal plate with parafilm and store inverted at 4°C.

  2. Isolate plasmid DNA with Miniprep ExpressTM.

  3. Identify recombinant clones by restriction enzyme analysis or PCR.

  4. Locate bacteria harboring the recombinant vectors from the number assigned on the master plate. Prepare stocks for storage. Prepare plasmid DNA in any desired scale (use table on pg 33 as a guide).


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