IGEM:MIT/2006/Notebook/2006-7-28

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Contents

Amazing News

Transformants on every plate!!!

To do (am)

  1. Miniprep 5 liquid cultures, make glycerols, and send DNA for sequencing -complete
  2. PCR pchBA -complete

pchBA PCR

  1. Lets try to amplify out pchBA together and seperately
  2. 3 test tubes + 1 control
  3. Dilute new Invitrogen pchBA primers into working stocks of 25μM
  4. Tube 1 (pchBA):
    • 49 μL PCR supermix
    • 1 μL pME3366 template (1 nanogram)
    • .6 μL pchB Forward (300 nanograms)
    • .6 μL pchA Reverse (300 nanograms)
  5. Tube 2 (pchB):
    • 49 μL PCR supermix
    • 1 μL pME3366 template
    • .6 μL pchB Forward
    • .6 μL pchB Reverse
  6. Tube 3 (pchA):
    • 49 μL PCR supermix
    • 1 μL pME3366 template
    • .6 μL pchA Forward
    • .6 μL pchA Reverse
  7. Run the tubes on the thermocycler at: 95deg for 3:00, then cycle through: (a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00

To do (pm)

  1. PCR cleanup pchBA PCR products and run 5 μL on a gel to see if successful and put in -20 fridge
  2. make 3 liquid cultures (A,B,C) from each (8) transformant plate and put in 37 room
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