IGEM:MIT/2007/Notebook/2007-7-16

=F2620 Expression Protocol= See characterization data for BBa_F2620. =Today's Agenda=
 * 1) Create Liquid Cultures in LB Media
 * 2) Dilute Back 1:500 in LB Media
 * 3) Grow up for an hour in warm room roller
 * 4) Split culture in two
 * 5) Add AHL to one culture to a final concentration of 1E-6M
 * 6) Grow up for 4 hours in warm room roller
 * 7) Spin Down, Decant, and Resuspend in PBS
 * 8) Measure absorbance at 600nm and red fluorescence level
 * Redo Overgrown Liquid Cultures
 * Send for Sequencing
 * Cut CPX and Backbone for ligation
 * Start designing high copy backbone
 * PCR off relevant portion of Mer operon from pto40 - Done
 * Organize fridge - Done
 * Design and order EC_20 Insert
 * Email Isadora + Tom Knight for Meeting Times--Toan--Done
 * Anneal FHUA Insert oligos - Done
 * Understand Meinke Inducible System

=Status of Expected Parts=
 * 1) IDT Primers + Inserts - Arriving Today--ARRIVED
 * 2) CPX - At FedEx Facility in Boston
 * 3) FhuA - ARRIVED

Sequencing of LC#1&2 from 3A

 * Miniprepped LC's of those colonies that grew only on Kan plates and not on Kan/Amp plates
 * Sent in 4 samples, one forward and one reverse for each LC
 * used 4 µl of DNA since each was approximately 30 ng/µl
 * Order Number 8046

Dilution of Ordered DNA

 * Made 100uM stocks of all IDT primers and single-stranded inserts
 * Diluted 100uM stocks of primers/inserts into working stocks of 10uM concentration

Annealing Complementary Primer Inserts

 * 10 uL of 10 uM of oligo
 * 2 oligos - total volume 20 uL
 * Mix with 80 uL water
 * PCR Thermocycler Temperatures
 * 95 C
 * Ramp down
 * 0.1 C/sec

Phosphorylation of Primer Inserts

 * 20 ul Annealed Oligo
 * 4 ul T4 DNA Ligase Buffer
 * 2 ul PNK (Kinase)
 * 14 H20
 * 37° - 1 hr, 65° - 20 mins

Digestion of FhuA backbones

 * used PCR Purification kit to purify backbone

Nanodrop of FhuA inserts/backbone

 * PICCS5 - 10 ng/ul
 * PHIE6 - 5 ng/ul
 * Insert- 650 ng/ul - instead just use rule of thumb .5 ul/20ng backbone

Ligation of FhuA inserts/backbone

 * Mix 1: PHIE6
 * .5 ul Ligase
 * 2 ul Ligation Buffer
 * 1.5 ul Insert
 * 10 ul Backbone
 * 6 ul H20
 * Mix 2: PICSS5
 * .5 ul Ligase
 * 2 ul Buffer
 * 1.5 ul Insert
 * 5 ul Backbone
 * 11 ul H20
 * Program 16IGEM