Team:Cambridge/Protocols/DNA Extraction from E.coli

Protocol Name

Preparation

Before using this protocol centrifuge 30ml cultured bactiera at 3000rpm for 15 minutes.

Theory

The miniprep kit relies on a column, which fits into a centrifuge tube which has different affinity for DNA depending on buffer which is washing through.

First centrifugation causes the cells to form a pellet at the bottom of the tube, this allows the medium in which the bacteria have been growing to be removed (the supernatant) and the cells can then be resuspended in another chemical. This prinicple is used in many protocols to allow the solution surrounding the cell replaced with various buffers in the kit.

Bacterial cells are lysed by exposing them to a detergent (SDS) which solubilises the cell membrane, whilst sodium hydroxide disrupts the cell wall and more importnantly denatures the genomic DNA, the plasmid DNA and the proteins.

The lysate is neutralized and adjusted to high salt concentration which causes denatured proteins, chromosomal DNA, cellular debris, and SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution.

The chromosomal DNA is separated from the plasmid DNA because the chromsomal DNA binds to the QIAGEN membrane in the column along with the precipitating proteins and debris, whilst the plasmid DNA stays in solution and can be removed in the supernatant.

Practice

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.

5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

6. Centrifuge for 30–60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.

This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5αTM do not require this additional wash step.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Safety

The safety implication of the procedure.