IGEM:MIT/2007/Notebook/2007-7-16: Difference between revisions
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*Cut CPX and Backbone for ligation | *Cut CPX and Backbone for ligation | ||
*Start designing high copy backbone | *Start designing high copy backbone | ||
*PCR off relevant portion of Mer operon from pto40 | *PCR off relevant portion of Mer operon from pto40 - Done | ||
*Organize fridge | *Organize fridge - Done | ||
*Design and order EC_20 Insert | *Design and order EC_20 Insert | ||
*Email Isadora + Tom Knight for Meeting Times--Toan--Done | *Email Isadora + Tom Knight for Meeting Times--Toan--Done | ||
*Anneal FHUA Insert oligos | *Anneal FHUA Insert oligos - Done | ||
*Understand Meinke Inducible System | |||
=Status of Expected Parts= | =Status of Expected Parts= | ||
Line 28: | Line 29: | ||
*Miniprepped LC's of those colonies that grew only on Kan plates and not on Kan/Amp plates | *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 | *Sent in 4 samples, one forward and one reverse for each LC | ||
*used | *used 4 µl of DNA since each was approximately 30 ng/µl | ||
*Order Number 8046 | *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
- Create Liquid Cultures in LB Media
- Dilute Back 1:500 in LB Media
- Grow up for an hour in warm room roller
- Split culture in two
- Add AHL to one culture to a final concentration of 1E-6M
- Grow up for 4 hours in warm room roller
- Spin Down, Decant, and Resuspend in PBS
- 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
- 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