Difference between revisions of "Eckdahl-replication-protocol"

Revision as of 16:26, 4 June 2014

Procedures

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

New Chaperone PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of amp+chlor)

- 0.4 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Gree

New Chaperone PCR Thermal Profile

- Initial denaturation: 10 minutes at 94 degrees

- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes

- Final extension: 74 degrees, 5 minutes

Determine origin with PCR; run on agarose gel

Origin PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.5 uL water

- 10 uL 2x GoTaq Green

Origin PCR Thermal Profile

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

Plamids

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

Origins

pSB1A2 High copy number

J119310 Low copy number