Team:RHIT/Protocol

Edit Here

Protocols

  • QIAprep Spin Miniprep Kit
    1. Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.
    2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
    3. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
    4. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
    5. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
    6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through.
    7. Recommended: Wash the QIAprep spin column by adding 500 µl Buffer PB. Centrifuge for 30-60 x and discard the flow-through.
    8. Wash the QIAprep spin column by adding 750 µl Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
    9. Centrifuge for 1 min to remove residual wash buffer.
    10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB or water to the center of the QIAprep spin column, let it stand for 1 min, and centrifuge for 1 min.

  • NEBuilder HiFi DNA Assembly Reaction Protocol
    1. Set up the following reaction on ice (for 2-3 fragment assembly):
      • 0.03 - 0.2 pmols Total DNA (recommended DNA ratio - vector:insert = 1:2) X µl
      • 10 µl NEBuilder HiFi DNA Assembly Master Mix
      • 10 - X µl deionized water
    2. Incubate samples in a thermocycler at 50ºC for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4-6 fragments are being assembled). Following incubation, store samples on ice or at -20ºC for subsequent transformation.
      Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
    3. Transform NEB 5-alpha Competent E. coli cells with 2 µl of the assembled product.

  • Boiling Prep Protocol

  • NEB High Efficiency Transformation Protocol

  • QIAquick Gel Extraction Kit

  • Ethanol Precipitation

  • Yeast Transformation (From Breeden Lab)