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Start Ligations

  • Began stage 2 ligations
    • (12D->24A)->6B
      • F2620 (6B) - Vector (Cut with S & P, use orange buffer)
      • C0261+I13504 (12D->24A) - Insert (Cut with X & P, use yellow buffer)
    • (7A->3O)->9G
      • R0062 (9G) - Vector (Cut with S & P, use orange buffer)
      • B0034+C0062 (7A->3O) - Insert (Cut with X & P, use yellow buffer)
  • Digests and gel running unsuccessful
  • No DNA was evident on the gel, thought to have been caused by incorrect maxi-prep of parts
  • Propose to re-maxiprep all parts that were maxi-preped yesterday on 4/8 Friday
  • PCR was also unsuccessful due to maxi-preping issues yesterday

PCR fusion Protein

  • Prepare on ice: Mix in a PCR tube the following:
    • 28.5 ul pure water
    • 5ul buffer (taq buffer containg sulphate)
    • 1ul of each primer
    • 10ul MgCl2 (from 25uM stock solution)
    • 2.5ul DNTP mix (10uM)
  • Dilute 1ul of maxiprep DNA nto 50ul water.
    • Take 1ul of this solution and add to reaction mixture.
    • Add 1ul of Taq Polymerase
  • Total Volume should be 50ul.
  • Program PCR machine
    • 94C for denaturation (1min)
    • Work out melting temperature from primer composition according to formula below. Also found on label of primer tube.
      • (2xA-T)plus(4xG-C)-5= Melting temperature.(1 min)
      • Calculate for both primers and use the lower of the two.
    • 72C for extension (1min-1min 15 sec depending on template size. 1min for 1KB template).
  • Run for 25 cycles
  • Leave at 4C after cycles (program machine).
  • Run DNA on gels to isolate.

Annealing Oligos

  • Oligos come as dry powder.
  • Resuspend in water (add 1ul water for every ug of powder)
  • Caution: careful when adding water - powdered disc may float out!
In a PCR tube:
  • Add 5ul each of the two complementary oligos
  • Add 2ul of O-buffer (orange top)
  • Make up the rest of the solution to 20ul with water
  • Place epindorf in PCR machine for 2-3 mins at 94C
  • Switch off machine and allow to cool for ~1hr to room temperature (oligos should anneal)
  • To add phosphates to the strands:
  • Mix 10ul of annealed oligos
  • 2ul of 10 times Buffer A
  • 1ul 10mM ATP
  • 6ul dH20
  • 1ul PNK
  • Leave in 37C waterbath for 30-60mins

Set up Maxi-Prep for 4/8

Cultured the following in 50 ug/mL Amp LB

  • 1I
  • 5M
  • 12D->24A colonies 1, 3, and 4
  • 16P
  • 7A->3O colony 2
  • 18I (electroporation successful)

Part I13207 Testing

  • Made up 10% L-arabinose solution
    • 1 g into 1 mL MilliQ
  • Made up 1M Zinc chloride solution
    • 1.363 g in 10 mL MilliQ
  • Protocol underway
  • Will be tested in 2 phases
    • Part 1 testing: Same as T9002 testing, can be done in parallel
    • Part 2 testing: To see on or off of AiiA with key parameters (arabinose, zinc chloride)

To Do on Friday

  • Maxiprep - 8:30 am
    • John C, Jimmy, Christin, Tom
  • Ligations - 10:30 am (dependent upon Maxiprep finishing)
    • John S, Jonny
  • PCR - 10:30 am (dependent upon Maxiprep finishing)
    • Deepti, Farah, Christin
  • Gel - 10:30 am (overnight PCR and maxipreps to check)
    • A few people who do maxiprep