Manual Qiagen PCR Purification

Overview of Manual Qiagen PCR Purification

Preparation:

• PM Buffer, PE Buffer, TE and a QiaQuick filter are used for this procedure. Check to be sure they are available in the Robot Room.
• The QiaQuick filters are placed on a "short" holder for all robot methods. Locate a "short" holder. These are usually found to the right of the sink. 
• TE is dispensed from a quarter holder. Locate the quarter holder and "tall" support. 
• Locate a drilled bottom 96 well plate to support the PCR plate coming off the thermocycler. 
• Connect the filtration flask and filter device to the vacuum pump on the Biomek. 
• Fill the QFill2 machine with PE and set the volume to 600 µl . Label an MJ Thermocycler plate "(Plate ID)PCR Frags (Today's Date)".

Procedure:

• PCRfrag1 will mix the PCR fragments created in the last method with PM Buffer and place that mixture in the QiaQuick filter. 
• The flexible non skirted PCR plate is thrown away and the QiaQuick filter is transferred to the Biomek. 
• PCR FRAG2rv will filter the PM buffer, 2 washes with PE and elute the PCR fragments with TE. 
• This method begins by turning the vacuum on and pulling the PM buffer through the QiaQuick filter. You are then required to take the QiaQuick filter to the QFill2 station for the addition of PE. PE will be added to the QiaQuick filter twice with a filtration step between each addition. 
• After the second filtration of PE, the Biomek will set up to elute the PCR fragments into the square deep well plate. 
• BRIEFLY centrifuge this square deep well plate to ensure all the liquid is at the bottom. 
• PCR TX will transfer the PCR fragments from the square deep well plate to the labeled MJ Thermocycler plate.
  
  
• Next Step: Picogreen Analysis

Manual PCR frag purification (PCRfrag manual)

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 
• QiaQuick plates are placed on "short" holders for robot methods 
• Fill QFill2 with PE and make sure its pressurized and filled

Method Sequence

• PCRfrag1-method mixes PB/PM solution with the PCR fragments and transfers the solution to the Qiagen QiaQuick plate.
• PCRfrag2rv-processes the PCR fragments which are eluted into a deepwell plate by the addition of 150 µl of TE. Prior to addition of TE, excess liquid from the plate drip defectors is removed by manual blotting of the plate on paper towels 
• PCR TX-method transfers plasmid solution from the deepwell plate to a thermocycler storage plate.

Multimek method PCRfrag1

Biomek method PCR FRAG2rv

Multimek method PCR TX