IGEM:MIT/2007/Notebook/2007-7-16: Difference between revisions

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(New page: =F2620 Expression Protocol= #Create Liquid Cultures in LB Media #Dilute Back 1:500 #Grow up for an hour #Spin Down and Resuspend in PBS #Add AHL)
 
 
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=F2620 Expression Protocol=
=F2620 Expression Protocol=
#Create Liquid Cultures in LB Media
#Create Liquid Cultures in LB Media
#Dilute Back 1:500
#Dilute Back 1:500 in LB Media
#Grow up for an hour
#Grow up for an hour in warm room roller
#Spin Down and Resuspend in PBS
#<font color=red>Split culture in two</font>
#Add AHL
#<font color=red>Add AHL to one culture to a final concentration of 1E-6M</font>
#<font color=red>Grow up for 4 hours in warm room roller</font>
#Spin Down, Decant, and Resuspend in PBS
#<font color=red>Measure absorbance at 600nm and red fluorescence level</font>
See [http://parts.mit.edu/registry/index.php/Part:BBa_F2620 characterization data] for BBa_F2620.
=Today's Agenda=
*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=
# IDT Primers + Inserts - Arriving Today--ARRIVED
# CPX - At FedEx Facility in Boston
# 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

Latest revision as of 11:47, 23 July 2007

F2620 Expression Protocol

  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

See characterization data for BBa_F2620.

Today's Agenda

  • 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
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