Team:BYU Provo/Team Thermosensor/Week8

Week 8 (June 5 - 11)

• 6/7
  • Added 6 ul loading dye to each PCR reaction tube and ran on gel @ 160 V for 17 min
    • Lane 1 - DNA ladder
    • Lane 2 - 24-1
    • Lane 3 - 24-2
    • Lane 4 - 24-3
  • It appears that lacZ amplified correctly, but pBAD did not for whatever reason, we had very little template left (PJG408) that could be part of the issue
• 6/6/11
  • Ligation for pBAD into pPLAT; put in tube labeled Lig 23-1
    • 6.5 ul H2O
    • 1.5 ul 10x ligase buffer
    • 1 ul T4 ligase
    • *1 ul vector (23-2)
    • *1 ul insert (23-1)
    • - Incubate for 30 min at room temp
      • Transformation
        • Followed Dr. Grose’s procedures. Incubated in 37 degree incubator overnight. - Addison
        • Back on ice @ 3:27
        • Take out of incubator @ 4:13
• 6/7
  • It appears that there is no cell growth on the plate labeled 6/6 23-1
  • Set up PCR for pBAD following volumes on prev. page
  • There is lacZ still in stock
  • Ran on “Phusion10” in Grose’s labe.
    • +25-1
      • pBAD recipe same as 24-1
    • We have pBAD!
  • PCR clean-up of pBAD
    • T26-1 PCRup w/ Dr. Grose
  • Low melt of T26-1 PCRup
    • Strong band, very nice looking
  • Cut out gel slice w/ Dr. Grose
  • pBAD GS T26-1
  • Ligation of pBAD w/ Dr/. Grose
    • Used insert: GS T26-1 and vector: GS T23-2
  • T26-1 pBAD + 23-2
    • Lig
    • 6.5 ul H2O
    • 1.5 ul 10x ligase buffer
    • 1 ul T4 DNA ligase
    • 3 ul vector
    • 3 ul insert
      • mix and sit for min
    • T26-1
      • Tran
        • T26-1 + 23-2
          • pBAD ligation transformed into E. coli DH5alpha
      • Plate
        • pBAD 26-1
        • pPLAT 23-2
        • Transformation
        • Placed in 37*C. If we have growth the next thing is colony PCR
        • We have growth!!
• 6/9/11
  • Colony PCR 26-1
    • Took 8 colonies from plate T26-1 and swirled them in 50 ul of H2O and then plated on “T26-1 lig” and placed the plate in 37*C overnight
    • Took the PCR tubes with 50 ul of H2O + the 8 colonies + boiled in thermocycle - this will then be used as template for the colony PCR
      • 19 ul of H2O
      • 2.5 ul Rxn buffer
      • 0.5 ul 10mM dNTPs
      • 0.5 ul IG11 (forward) primers
      • 0.5 ul IG 12 (reverse) primers
      • 0.5 ul Phusion DNA polymerase
      • 2 ul boiled colony sample

• • • Master Mix T26-1
      • 171 ul H2O
      • 22.5 ul 5x Rxn buffer
      • 4.5 ul 10 mM dNTPs
      • 4.5 ul IG11
      • 4.5 ul IG12
      • 4.5 ul Phusion polymerase
      • + add 2 ul boiled colony sample + 23.5 ul of master mix to each tube
        • Ran in Phusion 10
        • OOPS...USE Taq LIKE PROTOCOL CALLS FOR ON COLONY PCR...My bad
          • This was just the boiled template. I labeled the template and the PCR similarly, my fault...I will label the colony PCR as PCR next time + the template as “T” followed by a number.
          • Lanes 2-8=Tubes 1-8. No verification of insert. Will retry another colony PCR
        • Reused colonies 1-8 and boiled them from plate T-26-1
      • Master Mix A T26-A
        • 171 ul H2O
        • 22.5 ul 5x Rxn buffer
        • 4.5 ul 10 mM dNTPs
        • 4.5 ul IG11
        • 4.5 ul IG12
        • 4.5 Taq polymerase
        • add 2ul boiled colonies + 23.5 ul of this mix to each tube
      • Master Mix B T26-B
        • 171 ul H2O
        • 22.5 ul 5x Rxn buffer
        • 4.5 ul 10 mM dNTPs
        • 4.5 ul IG12
        • 4.5 ul IG38
        • 4.5 Taq polymerase
          • PCR: Ran 26-1 on a gel @165V for 18 min
            • It worked! It appears that we have pBAD in the pPLAT plasmid
              • Next step is to sequence the plasmid just to verify that it really worked and then we can put GFP and lacZ into the plasmid as well
              • No need to sequence...it’s a promoter