Manual Qiagen Plasmid Setup

1. Manual Qiagen Plasmid Setup

2. Overview of Manual Qiagen Plasmid Setup Setup Time: 30 minutes Runtime: ~20 minutes/plate Cleanup Time: 15 minutes • Preparation: • Check to be sure that there are enough experiment supplies (Resuspension Buffer, Lysis Buffer, and Neutralization Buffer, PE Buffer, TE, Turbofilter plates and QiaPrep plates) in the Robot Room. Resuspension Buffer is kept in the refrigerator of the robot room. Neutralization Buffer needs to be pre-chilled. This is done by placing it in the refrigerator of the robot room, at least 20 minutes prior to start. • Set up the filtration device and the vacuum pump on the Biomek, as illustrated in Biomek method HT96 Plasmid1rv. • Set up a paper towel for blotting, in Biomek slot B1. • Fill the QFill2 with PE and set the volume to 600 l. • Locate the “short” holders for the Turbofilter and QiaPrep plates. • You will need your pellet plate from “Manual Annealing Setup”, as well as a new deepwell plate for each pellet plate. • Label an MJ Thermocycler plate using the SAMI method “Print and Apply”. This plate should be labeled “(Plate ID)Plasmid (Today’s Date)”. • Procedure: • Multimek - BacResP1 will add resuspension buffer to the bacterial pellet in the square deep well plate from the “Manual Annealing Setup” process. • After addition of resuspension buffer, look to be sure pellets are resuspended in deepwell plates. Place the square deep well plate on the shaker to the left of the Multimek for 3-4 minutes. It may be necessary to “help” resuspend the pellets by GENTLY tapping the plate against the palm of your hand or foil seal the plate and vortex the samples. If you choose to vortex the samples, you will need to BRIEFLY centrifuge the plate to bring all the liquid to the bottom of the wells. • Multimek - Once the pellets are completely resuspended, PlasmidP1 will add lysis buffer to the square deep well plate and rinse the tips. Place plate on shaker next to Multimek for 30 seconds to 1 minute, to assist with lysis of cells. Do not allow the lysis reaction to carry on longer than 5 minutes. It is advisable to proceed one plate at a time from this step, until after method Plasmid2. • Multimek - Plasmid2 adds neutralization buffer to the square deep well plate, then transfers the solution to the Turbofilter plate. • The square deep well plate can be thrown out. The Turbofilter plate is transferred to slot B4 on the Biomek. The QiaPrep filter is placed in slot B3. A new square deep well plate is placed in slot B6 and the full reagent reservoir is placed in slot A4 with TE. • Biomek - HT96 Plasmid1rv purifies the plasmid DNA. The Turbofilter is filtered to remove the neutralization buffer. The QiaPrep filter is then washed twice with PE at the QFill2 station. The plasmid is eluted with TE into the new square deep well plate. Briefly centrifuge the deep well plate for 10 seconds at 500rpm before moving on to the next procedure. • Multimek - The plasmid is transferred from the square deep well plate to a labeled MJ Thermocycler plate for storage. This is done using PCR TX. • This plate is stored at 4ºC and is now ready to be used for transformations. • Next Step: Transformation

3. Multimek method BacResP1 After addition of ~ 30ml resuspension buffer, look to be sure pellets are resuspended in deepwell plates. Place deepwell plate on shaker next to Multimek for 3-4 minutes and tap plate against palm of hand to help with resuspension.

4. Multimek method PlasmidP1 After addition of ~ 30ml lysis buffer, place plate on shaker next to Multimek for 30 seconds to 1 minute, to assist with lysis of cells

5. Multimek method Plasmid2 After addition of ~ 35ml neutralization buffer, take QiaTurbo plate to Biomek for method HT96 Plasmid1rv

6. Biomek method HT96 Plasmid1rv After first transfer, discard QiaTurbo plate and if necessary blot the top of the QiaPrep plate with paper towel. The QiaPrep plate is washed at the QFill station. After the second wash step, begin cleaning the QFill station with water. Briefly centrifuge deepwell plate for 10 seconds at 500rpm before moving on to next procedure.

7. Multimek method PCR TX

8. Qiagen PlasmidSetup (LICRx HT.smt) • Before automation run – • Setup Biomek for Qiagen filtration method • Connect filtration flask to Biomek filter device and to vacuum pump • Turn on vacuum pump • Place blotting paper on position B1 of Biomek deck • Turbofilter and Qiaprep plates are placed on “short” thin plastic holders for robot methods • Fill QFill2 with PE and make sure its pressurized, filled, and set to 600 l/well • Method Sequence • BacResP1-method adds 120 l of resuspension buffer to the bacterial pellets. Plate is transferred to the shaker for approximately ten minutes to allow for resuspension of the pellet. • Plasmid1-transferes 250 l of lysis buffer to the resuspended pellet. The tips are rinsed with water to wash out any residual lysis buffer. • Plasmid2-method adds 350 lof resuspension buffer to the bacterial pellets and pipets up and down several times to mix. The solution is then transferred to a Turbofilter plate. • HT96Plasmidrv-method processed the bacterial lysate and elutes the purified plasmid with 150 l of TE. • PCR TX-method transfers plasmid solution from the deepwell plate to a thermocycler storage plate.