Eckdahl-replication-protocol

1. Grow up E. coli from stock overnight

a. One batch in amp, one in amp+chlor

b. 4 mM caffeine

2. 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.

3. Combine 0.5 mL of each clone.

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

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

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

b. 35 uL 40 mM caffeine solution per disk

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

d. 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.

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

6. Incubate upside down

a. 37degrees C overnight

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

7. 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.

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

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

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

11. Measure RFP and GFP fluorescence

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

13. Determine chaperone # with PCR; run on agarose gel

a. Mixture

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

c. primer cocktail with chaperone primers

d. water

e. 2x GoTaq Green

f. PCR Steps

g. Initial denaturation: 10 minutes at 94 degrees

h. 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes

i. Final extension: 74 degrees, 5 minutes

14. Determine origin with PCR; run on agarose gel

a. Mixture

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

c. 0.4 uL primer cocktail with origin primers 100 uM each

d. 7.6 uL water

e. 10 uL 2x GoTaq Green

f. PCR Steps

g. Initial denaturation: 94 degrees, 10 minutes

h. 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.

i. 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