Team:Debrecen-Hungary/protocols/miniprep

Scientific Background

This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium.

Overview

Please read the following notes before starting any of the QIAprep procedures.

Growth of bacterial cultures in tubes or flasks

1. Pick a single colony from a freshly streaked selective plate and inoculate a small culture of

1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for

12–16 h at 37°C with vigorous shaking.

Growth for more than 16 h is not recommended since cells begin to lyse and plasmid

yields may be reduced.(if the plasmid is low-growing we grow 16-18h) Use a tube or flask with a volume of at least 4 times

the volume of the culture.

2. Harvest the bacterial cells by centrifugation at > 8000 rpm (6800 x g) in a conventional,

table-top microcentrifuge for 10 min at room temperature (15–25°C).

The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5400 x g for

10 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge

tube until all medium has been drained.

Materials

Procedure

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. Pellet must homogene

Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

2. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix.

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 invert the tube immediately but gently 4–6 times.

To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.

4. Centrifuge for 20 min at 14,000 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. Must pipetting clean liquid.

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

Spinning for 60 seconds produces good results.

7. (Optional): 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 DH5α™ do not require this additional wash step.

Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.

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

Spinning for 60 seconds produces good results.

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. They are right about this.

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.

If you are concerned about the concentration of the DNA, you can alternatively add 30 μL water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases.

Notes & troubleshooting

Passing the lysate over the column twice increases yield by about 20%.

Contaminating salt from the initial lysate or the PB will ruin a sequencing reaction more frequently than eluting in the EB (10 mM Tris as a small component of the total sequencing reaction is negligible). I always elute with EB and my reactions sequence just dandy. There are two major sources of salt contamination: the inside upper edge of the spin column and the residual PB mixing with the PE wash. When you add the initial PE, it mixes with the leftover junk in the column. Spinning this through can only lower the salt to a level that was present after mixing. To get around these problems, I do two PE washes of about 300-500 μL. For the the first, I dispense the liquid from the pipette tip along the inner ledge of the spin column in a circular motion to wash off the residue there. I follow the first PE wash with a second to further de-salt the sample before the drying spin. Yes, it adds a step, but the time spend here is far less than waiting three days only to find out your sequencing didn't work.

Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields.

Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields

References

1. Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC382999/pdf/pnas00002-0073.pdf Proc. Natl. Acad. Sci]. USA 76, 615–619.

2. Birnboim, H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1522.

3. Sambrook, J. et al., eds. (1989) Molecular cloning: a laboratory manual. 2nd ed., [http://nhjy.hzau.edu.cn/kech/jycz/jczs/ml-introduction/Content.pdf Cold Spring Harbor Laboratory Press].

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