1 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
2 Mini-Preparation of Plasmid DNA Centrifugation - The process of separating substances by different densities by using a centrifuge. Supernatant Substances which are more dense settle to the bottom of the tube. Pellet the clump of cells and cellular debris found at the bottom of a centrifuge tube after centrifugation. Pellet Supernatant The nonpelleted solution left after centrifugation. Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (14,000 rpm) in the microcentrifuge.
3 Remove the growth medium by pouring out the supernatant. Gently tap the microcentrifuge tube on a paper towel to remove any residual supernatent. Resuspend the bacterial pellet in 200µl of solution I by pipetting up and down as 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 which break down DNA)
4 Add 200 µl of Solution II, invert gently 10 times. Do not vortex!! This will shear the DNA and contaminate your DNA preps. Solution 2 (alakaline lysis buffer): P2 is the solution that will lyse the cells. The buffer contains.2m NaOH and 1% SDS. SDS is a detergent that will destroy the bacterial membrane. NaOH will denature the chromosomal DNA and the plasmid DNA. Add 400 µl of Solution III. Invert gently 10 times. Do not vortex. Solution 3 (neutralization buffer): Solution 3 consists of 5M KOAc, ph 4.8 KOAc will readjust the ph and allow only the plasmid DNA to reanneal. Because the chromosomal DNA is so large, it cannot reanneal properly under these conditions.
5 Centrifuge for 5 minutes at full speed in the microcentrifuge. A white pellet will form on the bottom and side of the tube after centrifugation. 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.
6 Centrifuge for 1 minute at full speed, and drain the flow-through from the collection tube. Add 400 µl of Wash Buffer to the spin column contained in the 2 ml Collection Tube, centrifuge at full speed for 1 minute, and drain the flow through. Wash buffer Once the plasmid DNA has been added to the column, the wash buffer is used to wash away any unwanted material.
7 Place the spin column in a fresh 1.7 ml microcentrifuge tube and centrifuge again for 1 minute at full speed to remove any residual wash solution that might still be in the column. Place the spin column into an appropriately labeled 1.7 ml microcentrifuge tube and add 60 µl of EB (Elution buffer) to the column. Elute - Remove plasmid DNA from the column 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 H 2 O can also be used to elute the DNA.
8 Remove the spin column from the labeled 1.7 ml microcentrifuge tube and close the lid on the tube tightly. 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 Book keeping!! Enter the clone name and prep date into Google Docs