Manual Qiagen Plasmid Setup

Overview of Manual Qiagen Plasmid Setup

Preparation:

• Check to be sure there is enough Resuspension Buffer, Lysis Buffer, and Neutralization Buffer, PE Buffer, 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. 
• Set up filtration device and the vacuum pump on the Biomek 
• Fill the QFill2 with PE and set the volume to 600 µl. 
• Locate the "short" holders for the Turbofilter and QiaPrep plates. 
• You will need your pellet plate from "Manual Annealing Setup". 
• Label an MJ Thermocycler plate using the SAMI method "Print and Apply". This plate should be labeled "(Plate ID) Plasmid (Today's Date)".

Procedure:

• BacRes1 will add resuspension buffer to the bacterial pellet in the square deep well plate from the "Manual Annealing Setup" process. 
• Place the square deep well plate on the shaker to the left of the Multimek for 10-20 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.
• Once the pellets are completely resuspended, PlasmidP1 will add lysis buffer to the square deep well plate and rinse the tips. Do not allow the lysis reaction to carry on longer than 5 minutes. 
• 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 place 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. 
• HT96 Plasmid1rv purifies the plasmid DNA. The Turbofilter is filtered to remove the neutralization buffer. It is then washed twice with PE at the QFill2 station. The plasmid is eluted with TE into the new square deep well plate. 
• 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

LIC Reaction Setup (LICRx HT.smt)

Before automation run -

Method Sequence

• BacResP1-method adds 120 µl 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 µl of lysis buffer to the resuspended pellet. The tips are rinsed with water to wash out any residual lysis buffer. 
• Plasmid2-method adds 350 µl of 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 µl of TE. 
• PCR TX-method transfers plasmid solution from the deepwell plate to a thermocycler storage plate.

Multimek method BacResP1

Multimek method PlasmidP1

Multimek method PlasmidP2

Biomek method HT96 Plasmid1rv

Multimek method PCR TX