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now: Fri Sep 17 05:41:31 2021 ... mod: Sun Apr 4 18:35:58 2021
Plasmid Maxipreps using the Qiagen Kit
Use the Qiagen Plasmid Maxi prep kit to make typical stocks of plasmids. The maxi prep kit uses affinity columns that have a listed maximum capacity of 500 μg of DNA, although they are actually capable of binding more like 800 μg. It is important not to start with too many cells, otherwise the kit will not work efficiently. For high copy number plasmids (most modern plasmids are high copy number) start with a 200 ml overnight bacterial culture grown in LuriaBroth
Mostly follow the protocol in the Qiagen Plasmid Purification Handbook, with several notable exceptions. The protocol is reprised below.
- Start with a 200 ml overnight bacterial culture grown in LuriaBroth. Make a GlycerolStock by mixing 300μl of 50% glycerol with 700μl of the culture and store at -80C.
- transfer the remaining cells to a 250 ml centrifuge tube and pellet cells using a GSA rotor, 6000 rpm (approximately 6000 g) for 10 minutes at 4C. Then discard the supernatant.
- the pellet can be frozen indefinitely at -20C if desired, or stored on ice for several hours
- Resuspend the bacterial pellet in 10 ml Buffer P1 (this solution is stored at 4C; be sure the RNAse has been added to the P1) and transfer the resuspended cells to a 50 ml polypropylene (translucent) oak ridge tube
- be sure the pellet is completely and homogeneously resuspended leaving no chunks of cells. an easy way to do this is to pipette the Buffer P1 up and down over the pellet using a 10 ml pipette until the cells are completely dispersed. vortexing the cells causes a lot of splashing, and does not do as good a job of breaking up the cell pellet
- Add 10 ml Buffer P2 to the resuspended cells, and mix gently, but thoroughly. Incubate at room temperature for 5 minutes.
- this is alkaline lysis with SDS and NaOH. the solution will become quite gloppy with the addition of Buffer P2. invert the tube multiple times to mix. it is also helpful to rotate the tube around to disperse the material over the sides of the tube. do NOT let the resuspended cells stay in Buffer P2 for longer than 5 minutes; the goal is to have the cloudy bacterial solution be homogeneously cleared. if the solution clears sooner than 5 minutes, proceed directly to the next step. overtreatment with SDS and NaOH can irreversibly denature the plasmid DNA
- Add 10 ml Buffer P3 to the solution. mix immediately and completely by vigorous shaking, then incubate on ice for 20 minutes
- this solution neutralizes the NaOH from the previous step and precipitates the SDS by addition of potassium ions. the published protocol says to "mix immediately but gently by inverting 4-6 times". the published protocol is wrong. very vigorous shaking is needed to efficiently mix Buffer P3 with the gloppy solution that resulted from the addition of P2 to completely precipitate the SDS (but do not vortex). if the SDS is not completely precipitated at this step, it will precipitate later in the column, plugging the column and ruining the prep. not mixing Buffer P3 thoroughly enough is the most frequent cause of failure of the maxiprep protocol
- Centrifuge at 30000 g for 20 minutes at 4C. while centrifuging equilibrate columns (next step)
- 16000 rpm in an SS34 rotor or 14500 rpm in an SA-600 rotor
- Set up one column (500 μg capacity) for each 200 ml bacterial culture. Add 10 ml Buffer QBT to equilibrate the column and allow the QBT to drain completely from the column
- it is ok to let the column "run dry" since the upper frit will protect the actual column matrix
- Add the supernatant from the above centrifugation step to the equilibrated column and allow it to flow through the column completely
- a second spin is unnecessary. avoid allowing a lot of precipitate to enter the column (a little is ok though). if there is loose precipitate on top of the supernatant after the first spin, it can be removed by pushing a plastic pipette tip (1 ml "blue tip") through it - the precipitate will stick to the outside surface of the pipette tip and can be discarded
- if the column become plugged, it means there was insufficient mixing with Buffer P3. start the prep again if this happens and next time really shake the solution after adding Buffer P3
- Add 30 ml Buffer QC to the column to wash the matrix, allowing the QC to flow through the column completely
- 30 ml is approximately the volume of the column. you don't have a measure out 30 ml, just pour in the buffer until the column is nearly filled
- Repeat the column wash with a second treatment with 30 ml Buffer QC, allowing complete flow through
- Transfer the column to a 50 ml polycarbonate collection tube. Add 15 ml Buffer QF to elute the plasmid DNA from the column
- technically, the polycarbonate is not resistant to the subsequent alcohol treatment. In practice, polycarbonate works fine and has the advantage of being transparent
- Precipitate the plasmid DNA by adding 10.5 ml (0.7 volumes) room-temperature isopropanol. First mix thoroughly by repeated gentle inversion, then shake vigorously to fully mix. Let stand two minutes to allow the small bubbles to clear. Centrifuge at 30000 g for 20 minutes at 4C
- it is helpful to mark the outer side of the centrifuge tube to aid in locating the plasmid pellet. the plasmid will either pellet at the bottom of the tube, or will occasionally form a streak of precipitate up the side of the tube (due to the presence of small air bubbles in the solution)
- Carefully discard the supernatant and set the tubes upright on an angle to allow the isopropanol to drain from the walls to the bottom of the tube off of the pellet. When the isopropanol has drained to the bottom, aspirate it with a vacuum line, or pipette, taking care not to aspirate the pellet.
- Note where the pellet is in each tube immediately after the centrifugation stops. Be careful not to unnecessarily agitate the tube as the pellet does not adhere very tightly to the walls of the tube. Decant the supernatant immediately after centrifugation as the pellet will start to detach from the tube wall with time. → THIS IS REALLY IMPORTANT It may be helpful to decant the supernatant into a clean beaker so that the pellet can be recovered in the event that it detaches from the bottom of the tube.
- Resuspend the pellet in 800 μl TE. First allow the TE to sit on the pellet for a few minutes to hydrate the DNA, then complete the resuspension by gentle repeated pipetting. If the DNA is in a streak on the side of the tube, it can be recovered by repeatedly pipetting the TE over it. Transfer the resuspened DNA to a 1.5 ml eppendorf tube
- the published protocol calls for a 70% ethanol wash at this point. do not do this - it is very easy to lose the pellet if you do
- Add 40 μl 8M LiCl to the resuspended DNA and vortex to mix
- this is half of the standard amount of salt added to an EthanolPrecipitation reaction. there is enough carryover salt from the column elution to make adding more unnecessary. the goal of this second precipitation is removal of that carryover salt
- Divide the TE/LiCl solution equally into two 1.5 ml eppendorf tubes (440 μl per tube), and add 2 volumes of room temperature 100% ethanol (800 μl ethanol added to each tube). mix by vortexing
- the plasmid DNA should immediately reprecipitate
- microfuge for 2 minutes to pellet the plasmid DNA, discard the supernatant, rinse the pellets with 500 μl 75% ethanol, discard the rinse ethanol, microfuge briefly to recover rinse ethanol from the sides of the tubes and remove residual ethanol with a pipette tip.
- add 250 μl 1/10x TE or H20 to each pellet. allow to rehydrate/dissolve overnight at room temperature
- it is not possible to dissolve the DNA both thoroughly and quickly. do not try to use or quantitate the DNA the day you do the plasmid prep -- the DNA will resuspend, but will not actually completely dissolve
- the next day, mix the hydrated DNA by vortexing until completely dissolved
- microfuge the DNA to pellet the fines from the column, remove, combine and save the plasmid supernatants in a new 1.5 ml eppendorf tube. quantitate by taking an the A260 reading. store at -20C
- if necessary, the prep can be temporarily halted between the two Buffer QC wash steps. you can, for example, put on the first QC wash, then go to lunch, then when you come back do the second Buffer QC wash