|Line 47:||Line 47:|
Revision as of 21:13, 15 May 2007
For purifying plasmid DNA from Escherichia coli cells, the Qiagen Spin Miniprep Kit produces quite reliable results.
Buffer P1 (in case you need to make it yourself) is:
- 50 mM Tris-HCl pH 8.0
- 10 mM EDTA
- 10 μg/ml RNaseA
The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).
See here or here for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit.
Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge
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. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37. Please read “Important Notes” on pages 19–21 before starting. Note: All protocol steps should be carried out at room temperature.
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) 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.
- 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.
- 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.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
A compact white pellet will form.
- Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 s. Discard the flow-through.
Spinning for 60 seconds produces good results.
- (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.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
Spinning for 60 seconds produces good results.
- 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.
- 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. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.
- If you are doing more than ~10 minipreps simultaneously, it can save time to switch to the vacuum manifold version of this protocol since you eliminate having to load and unload samples into the centrifuge.
- The sequencing center has begun using new machines and as a result you may want to consider eluting in water rather than EB. See note from sequencing center.
The elution is dependent on pH, however measuring the pH of unbuffered water is difficult. However, anecdotally we have been able to get good yields using the water from the stock room. Eluting in deionized water from the Knight lab has also produced good results.
- I use the "mini-fuge" for the binding and washing steps. You still have to do the drying step after the PE wash in a "real" microfuge though.
- 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.