Protocol for Preparing DNA Block

Detailed Protocol

2. Dilute the sample

• We use a 5 ng/µL dilution for our samples. For the master block, we prepare a total volume of 500 µL for each sample (half will go into the left side of the plate and half will go into the right side of the plate).
  
  
• Calculate the dilutions (easily done in an Excel spreadsheet). Make a copy of this spreadsheet and file it (along with notes on when the block was made, by whom and any other important details) in the blue "PGA DNA info" folder.
  
  
• Use autoclaved ddH2O and aerosol barrier pipette tips to prepare the diluted samples. Keep the DNA on ice as much as possible during this step.
  
  
• Calculate the dilutions (easily done in an Excel spreadsheet). Make a copy of this spreadsheet and file it (along with notes on when the block was make, by whom, and any other important details) in the blue "PGA DNA Info" folder.
  
  

3. Pipette the DNA samples into the deep-well plate

• Label the plate with the date, the DNA sample order listing (e.g., D001-D008, D009-D016; put this information on the side of the plate), and the concentration (5 ng/µL).
  
  
• Using the table above as a template, add the full volume of each sample to its spot on the LEFT side of the block. Keep the block on ice as much as possible while this is done.
  
  
• Use a multichanel pipette to move half of the volume of each column over to the corresponding column on the RIGHT side of the block.
  

4. Cover with oil and a mat; refrigerate

• Cover the samples with enough oil (about 100 µL) so they are totally covered.
  
  
• Put the mat on the block and store in the refrigerator.
  
  

V. Protocol for setting up the DNA working block

By Hand:

• Label another deep-well plate with the date, DNA concentration, and the names of the DNA samples.
  
  
• Using a multichannel pipette and barrier tips, transfer 150 µL from the master block to the corresponding wells of the working block. Do not cover these samples with oil; oil is not needed, since the samples are used within a week and thus there is little evaporation. File notes about the date of the procedure, who did it, and any other important details in the blue "PGA DNA Info" folder.
  
  
• If air appears in the pipette tips, the working block DNA supply is too low for pipetting. At this point, start a new working block rather than add DNA to the low working block. Be sure to record in the "PGA DNA Info" folder when the new working blocks are made.
  

By Robot: