Manual Annealing Reaction Setup

Overview of Manual Annealing

Preparation:

• Take competent cells (XL-1) and vector plates out of the freezer to thaw. Place them on top of the ice in an ice bucket. You will need about 5mL of competent cells per plate.  Take competent cells (XL-1) and vector plates out of the freezer to thaw. Place them on top of the ice in an ice bucket. You will need about 5mL of competent cells per plate. 
• Prepare the thermal chiller/exchanger and place on slot A3 of the Biomek. 
• Turn the heat block in the robot room on to the low setting, this should be at 48ºC. 
• Prepare 2xYT/Amp or Amp/LB/Glucose media (~100ml/plate).[40% Glucose located in labroom #1]. This will be used to prepare the LB/Amp reservoir. 
• Prepare LB/Amp and fill the QFill2 station. You will need about 100mL per plate of LB/Amp. This will be used to prepare a square deepwell plate with 1mL/well of LB/Amp.

Procedure:

• Anneal1 will mix a portion of the labeled LIC plate with the vector you've taken from the freezer and thawed. 
• This plate will sit at room temperature for 5-20 minutes to allow enough time for the annealing reaction. 
• Anneal2HT will add 45 µl of XL-1 competent cells to the plate from step 1. 
• BRIEFLY centrifuge this plate to be sure the competent cells mix with the annealing reaction and incubate this plate at 4ºC (usually on a ice) for 5-15 minutes. 
• Heat shock the cells by placing the plate on the heat block set to 48ºC for one minute. 
• The plate is then transferred back to 4ºC (usually on a ice) for 2-10 minutes before adding LB. 
• Anneal5 will add 120 µl of LB to the transformation plate. After the addition of LB the plates are incubated at 37ºC for 30 minutes. 
• Anneal4 transfers 20 µl of the culture from step 5 to a square deep well plate for overnight growth in a shaking incubator at 37ºC.
• Allow the culture to grow for 18-20 hours (usually overnight). In the morning, centrifuge the plate for 20 minutes at 3,000rpm and dump off the supernatant. 
• The pellets may be used immediately or stored at -20ºC until processed in the next procedure.
  
  
• Next Step: Manual Qiagen Plasmid Setup

Manual Annealing Reaction Setup

Before automation run -

Method Sequence

• Anneal1-method aspirates 4 µl of LIC reaction and mixes with 4 µl of pre-arrayed vector. Plates are stored at room temperature until the next method step. 
• Anneal2HT-arrays 45 µl of XL-1 competent cells into the annealing plate. 
• Anneal5-method transfers 120 µl of LB to transformation plate. Cultures are incubated at 37oC to enable recovery of antibiotic resistance. 
• Anneal4-method transfers 20-30 µl of recovery culture to a deepwell growth plate. Transfer volume is typically 20 µl but may be adjusted higher.

Multimek method Anneal1

Biomek method Anneal2HT

Multimek method Anneal5

Multimek method Anneal4