Difference between revisions of "Eckdahl-replication-protocol"
(Created page with "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 t...") |
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| − | + | ==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 | |
| − | + | <b>Mixture</b> | |
| − | + | 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone), | |
| + | primer cocktail with chaperone primers | ||
| − | + | water | |
| − | + | 2x GoTaq Green | |
| − | + | <b>PCR Steps</b> | |
| − | + | - Initial denaturation: 10 minutes at 94 degrees | |
| − | + | - 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes | |
| − | + | - Final extension: 74 degrees, 5 minutes | |
| − | + | Determine origin with PCR; run on agarose gel | |
| − | + | <b>Mixture</b> | |
| − | + | - 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone), | |
| − | + | - 0.4 uL primer cocktail with origin primers 100 uM each | |
| − | + | - 7.6 uL water | |
| − | + | - 10 uL 2x GoTaq Green | |
| − | + | <b>PCR Steps</b> | |
| − | + | - 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. | |
| + | |||
| + | - Final extension: 74 degrees for 5 minutes | ||
| + | |||
| + | ==Plamids== | ||
| + | |||
| + | <b>J119346</b> | ||
– High promoter | – High promoter | ||
| Line 91: | Line 98: | ||
– RFP | – RFP | ||
| − | + | <b>J119347</b> | |
– Low promoter | – Low promoter | ||
| Line 99: | Line 106: | ||
– GFP | – GFP | ||
| − | Origins | + | == Origins == |
| − | pSB1A2 High copy number | + | <b>pSB1A2</b> High copy number |
| − | J119310 Low copy number | + | <b>J119310</b> Low copy number |
Revision as of 18:01, 21 May 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
Mixture
2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone), primer cocktail with chaperone primers
water
2x GoTaq Green
PCR Steps
- Initial denaturation: 10 minutes at 94 degrees
- 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes
- Final extension: 74 degrees, 5 minutes
Determine origin with PCR; run on agarose gel
Mixture
- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),
- 0.4 uL primer cocktail with origin primers 100 uM each
- 7.6 uL water
- 10 uL 2x GoTaq Green
PCR Steps
- 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.
- 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
