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

Amazing News

Transformants on every plate!!!

To do (am)

1. Miniprep 5 liquid cultures and sent 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 4 liquid cultures (A,B,C,D) from each (8) transformant plate and put in 37 room