You are not logged in.
now: Wed Oct 18 21:35:33 2017 ... mod: Wed Dec 9 12:05:43 2015
Log in
QiagenPlasmidMaxiprep
QiagenPlasmidMaxiprep

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.

  1. 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.
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. Repeat the column wash with a second treatment with 30 ml Buffer QC, allowing complete flow through
  11. 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
  12. 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)
  13. 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.
  14. 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
  15. 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
  16. 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
  17. 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.
  18. 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
  19. the next day, mix the hydrated DNA by vortexing until completely dissolved
  20. 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

Comments

Bueno, la cosa se va calentando y al mismo tiepmo personalizando TCpace.. aunque no estoy de acuerdo contigo en muchas cosas creo que si, sois humanos, evidentemente.. pero el protocolo de emergencia, segun decis es algo para lo que se os prepara y teneis que estar mentalizados en que en cualquier momento es necesaria vuestra intervencif3n!!! y teneis que estar al 100% porque sois los que mejor conoceis esta situacif3n, las formas de actuacif3n y.. en definitiva, al avif3n, el procedimiento.. las llamadas!!!.. teneis que pensar que nosotros en el cielo estamos en tierra hostil y vosotros teneis que esta ahed para apoyarnos, para hacernos todo mas agradable y si en muchos momentos no teneis sangre fria.. transmitis muchedsima desconfianza.. leer que sois humanos y a lo mejor por eso tus compaf1eros sufrieron un shock no es algo que a una persona somo a mi (con cierto respeto a volar) ayude.. mas bien todo lo contrario!! hombre, que si os pasa eso en el momento que hay un accidente, la liamos parda, no??.. no se si teneis examen con apuntes o no, y es algo que realmente me da igual pero lo que si me gustaria exigir es que la persona de la que en ciertos momentos del vuelo dependo, tenga la suficiente fuerza psicolf3gica como para actuar como en teoria os ensef1an af1o tras af1o y sinceramente, esto no lo digo con intencif3n de meter el dedo en la herida, ni con la intencif3n de que la gente opine sobre ello pero poneros un poco en nuestro lugar en el lugar de la esposa del fallecido si, tus compaf1eros supongo que estare1n pasando un infierno por lo vivido, pero no menos que el resto de pasajeros no menos que las enfermeras que vieron que tardaba alguien en ofrecerse a relevarlas, no menos que la chica que se prestf3 a ayudar mientras ellos estaban paralizados y por supuesto,,, muuucho menos que el fallecido y que su esposa. Lo que tambien quiero hacer es darte las gracias por comentarlo todo sintiendote implicada y amenazada, pero manterniendo en todo momento la educacif3n al explicarnos las cosas. GRACIASTCPFR . Me parece un error el haber desviado el tema del objetivo inicial pero ya que todos opinamos de Ryanair.. no quiero ser menos bfAcaso en POR LO MENOS una Comunidad autf3noma no ha amenazado tu compaf1ia con que se iba si quitaban las subvenciones? y no me lo ha contado el amigo de un amigo de un amigo bfEres de las personas que cree que los pasajeros son respetados POR Ryanair? a pesar de que sea la empresa que te da de comer, espero que solo pienses eso de cara al publico o tu no conoces casos de una amiga de una amiga de una amiga que se sienta como un numero volando con esa CIA?.. Y que conste que yo nunca he tenido problemas con tus compaf1eros pero he visto y vivido muchas situaciones bfPorque es imposible conseguir que te envien la factura de tus vuelos en menos de 5 meses?.. bfno los tienen declarados todos?.. he decidido dejar de volar con vosotros porque no podia presentar las facturas a mi empresa Es evidente que Ryanair es una empresa low cost.. pero por ello no tiene que tratarnos como billetes andantes, numeros!! y es lo que hacen no quiero convertir esto en un debate sobre la adecuacif3n o no de Ryanair pero tampoco quiero que se nos tome por tontos!! . hay otras compaf1ias low cost en las que el trato al pasajero es mucho mejor, y no por ello los billetes son mucho mas caros.. y lo a gustito qe viajas a Barcelona a pesar de que tu jefe se queje constantemente porque le moleste que le tuteen Y que conste, que mi ultimo billete de Ryanair, en Septiembre, me costf3 i/v algo mas de 160 € sin flexibilidad horaria no es tan barata, no??..Y no espero respuesta a mis preguntas solo ESPERO QUE CON ESTO SE ACABE el hablar sobre lo buenediiiima que es la compaf1ia, lo bien que nos tratan para lo barata que es EL OBJETIVO de todo esto he entendido que no es ese.. que es evitar que vuelva a pasar lo que ha ocurrido haciendo que todas las compaf1ias pongan algo de su parte, ya sea en preparacif3n del personal o en medios por lo menor intentarlo!!! Seluvega cuenta con nosotros!! ya nos iras contando Saludos y, por favor, disculpas a los aludidos.
edit this text