DNA concentration normalisation

Katherine Jordan

Qiagility robot

QIAgility Robot Protocol for Normalizing DNA

  • Picogreen all DNA, and keep concentrations for robot. Save file with concentrations in .csv file

    • File needs to be A1, B1, C1, D1, …, H1, A2, B2, …, H2, …, A12, …, H12

    • Can keep fluorescence scores to do calculations on .csv file

    • File template on thumb drive, in DNA Quant folder

  • Centrifuge DNA plate for 2-3 minutes

  • Load thumb drive into computer beside robot

  • Load original DNA plate C1, New plate (labeled) C2, autoclaved double distilled H20 in top left circle of the robot

  • QIAgility software  Open

    • File  Open  Desktop  Yuye Barcode

    • Highlight C1 (original DNA plate) (-) in top right corner  Qiagen 96 well Micro  OK

    • Highlight C2 (new normalized DNA plate) (-) in top right corner  Half skirted

    • DNA plate info (far right table)  highlight C1  Import

    • Import file from thumb drive (location)

      • Start import from row  1 (depends on file)

      • Load concentration from column  7 (column with concentration from csv file)

    • Click Import  Finish

    • Highlight C2  Reaction list (top right table)

      • Final volume  10ul (desired volume)

      • Final concentration  10ng/ul (desired concentration)  OK

    • Click Green arrow run button  Save data

    • Make sure no error messages present (all writing is blue)

    • If it has less than 1ul to pipet you will need to change the concentration by clicking on the C1 plate and individually changing the concentration on that well

    • Run program… approx. 35 minutes

  • Take DNA plates, make sure enough water is left, put top back on it

  • Exit out of software

Pool DNA after ligation

Pool_96 protocol

  • Put 20ul of water into round bottom of 1.7ml centrifuge tube

  • Place this tube in bottom (waste) left corner of far left block

  • C2 reaction tab -> make sure 5ul of each sample is taken from C1 (plate) and put in waste

  • Check that C1 is the correct plate

  • No warnings present and check everything

  • Start