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DNA mini preparation

Mini-Preparation of Plasmid DNA• Mini-Prep = The isolation and purification of plasmid DNA from

bacteria.• In order to use a vector for cloning, sequencing, etc., it is

necessary to isolate the vector in a highly purified form.• Many companies now sell “kits” which provide all the solutions

necessary for preparing DNA.• Most Mini-Preparation techniques use the alkaline lysis

protocol.

Mini-prep

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Mini-Preparation of Plasmid DNA• Centrifugation - The

process of separatingsubstances by differentdensities by using acentrifuge.– Substances which are more

dense settle to the bottom ofthe tube.

• Pellet – the clump of cellsand cellular debris found atthe bottom of a centrifugetube after centrifugation.

• Supernatant – Thenonpelleted solution leftafter centrifugation.

Supernatant

Pellet

Mini-Prep Protocol

• Transfer 1.5 ml of theculture to a microfugetube and pellet thecells for 1 minute atfull speed (14,000rpm) in themicrocentrifuge.

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Mini-Prep Protocol

• Remove the growthmedium by pouringout the supernatant.

• Gently tap themicrocentrifuge tubeon a paper towel toremove any residualsupernatent.

Mini-Prep Protocol

• Resuspend the bacterial pellet in 200µlof solution I by pipetting up and downas well as vortexing.

• Solution I (resuspension buffer)– Solution I contains three components:

glucose, Tris and EDTA.– Glucose and Tris are used to buffer the pH

of the cell suspension.– EDTA chelates divalent cations (ions with

charges of +2) such as Mg++.– This helps break down the cell membrane

and inactivate DNAase (enzymes whichbreak down DNA)

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Mini-Prep Protocol

• Add 200 µl of Solution II, invert gently10 times. Do not vortex!! This willshear the DNA and contaminate yourDNA preps.

• Solution 2 (alakaline lysis buffer):P2 is the solution that will lyse thecells. The buffer contains .2M NaOHand 1% SDS.– SDS is a detergent that will destroy

the bacterial membrane.– NaOH will denature the

chromosomal DNA and the plasmidDNA.

Mini-Prep Protocol• Add 400 µl of Solution III. Invert gently

10 times. Do not vortex.• Solution 3 (neutralization buffer):

Solution 3 consists of 5M KOAc, pH4.8– KOAc will readjust the pH and allow

only the plasmid DNA to reanneal.– Because the chromosomal DNA is

so large, it cannot reanneal properlyunder these conditions.

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Mini-Prep Protocol

• Centrifuge for 5 minutes atfull speed in themicrocentrifuge.

• A white pellet will form on thebottom and side of the tubeafter centrifugation.

Mini-Prep Protocol• The plasmid DNA must now be purified away from the rest of

the cytoplasm and concentrated.• Affinity chromatography

– DNA selectively binds to the column.• Pour the supernatant into the appropriately labeled spin

column which has been inserted into a 2 ml collection tube.

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Mini-Prep Protocol

• Centrifuge for 1 minute at fullspeed, and drain the flow-throughfrom the collection tube.

Mini-Prep Protocol

• Add 400 µl of Wash Buffer to the spincolumn contained in the 2 ml CollectionTube, centrifuge at full speed for 1minute, and drain the flow through.

• Wash buffer– Once the plasmid DNA has been added to

the column, the wash buffer is used towash away any unwanted material.

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Mini-Prep Protocol

• Place the spin column in a fresh 1.7 mlmicrocentrifuge tube and centrifugeagain for 1 minute at full speed toremove any residual wash solution thatmight still be in the column.

Mini-Prep Protocol• Place the spin column into an appropriately

labeled 1.7 ml microcentrifuge tube andadd 60 µl of EB (Elution buffer) to thecolumn.

• Elute - Remove plasmid DNA from thecolumn

• Centrifuge at full speed for 1 minute.• Elution buffer

– EB removes the plasmid DNA from the column.– It is important to keep this volume relatively small in

order to have a reasonable DNA concentration.– Autoclaved H2O can also be used to elute the DNA.

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Mini-Prep Protocol• Remove the spin column from the labeled 1.7 ml

microcentrifuge tube and close the lid on the tubetightly.– Store it in a freezer box with your school number and your initials.

• Place the freezer box in the freezer.• Enter the name and location of the plasmid in the clone log

Enter the clone name and prep date into Google Docs

Book keeping!!