IGEM:IMPERIAL/2006/LabCalendar/2006-8-3

• IGEM:IMPERIAL/2006/LabCalendar/2006-8-3 Events
• Day in the lab
• Project Progress

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