Difference between revisions of "Eckdahl-replication-protocol"

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(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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1. Grow up E. coli from stock overnight
+
==Procedures==
  
−
a. One batch in amp, one in amp+chlor
+
Grow up E. coli from stock overnight
  
−
b. 4 mM caffeine
+
- One batch in amp, one in amp+chlor
  
−
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.
+
- 4 mM caffeine
  
−
3. Combine 0.5 mL of each clone.
+
Prepare stock of .25 g/mL L-arabinose
  
−
4. Spread 50 uL with 15-20 beads onto Tet-only LB plates
+
- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL
  
−
5. Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)
+
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.
  
−
a. Commercial disks (need to have a certain thickness to absorb enough solution)
+
Combine 0.5 mL of each clone.
  
−
b. 35 uL 40 mM caffeine solution per disk
+
Spread 50 uL with 15-20 beads onto Tet-only LB plates
  
−
c. Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute
+
- Prepare tetracycline-LB according to MWSU bacterial media protocol.
  
−
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.
+
- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)
  
−
e. Let sit for 5 minutes with lid cracked open under sterile hood
+
- Commercial disks (need to have a certain thickness to absorb enough solution)
  
−
6. Incubate upside down
+
- 35 uL 40 mM caffeine solution per disk
  
−
a. 37degrees C overnight
+
- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute
  
−
b. Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)
+
- 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.
  
−
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.
+
- Let sit for 5 minutes with lid cracked open under sterile hood
  
−
8. Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.
+
- Incubate upside down
  
−
9. Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.
+
- 37degrees C overnight
  
−
10. Incubate and measure A590 to determine how many colonies are still growing.
+
- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)
  
−
11. Measure RFP and GFP fluorescence
+
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.
  
−
12. Spot each colony on a single plate (2 uL per spot)
+
Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.
  
−
13. Determine chaperone # with PCR; run on agarose gel
+
Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.
  
−
a. Mixture
+
Incubate and measure A590 to determine how many colonies are still growing.
  
−
b. 2 uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),
+
Measure RFP and GFP fluorescence
  
−
c. primer cocktail with chaperone primers
+
Spot each colony on a single plate (2 uL per spot)
  
−
d. water
+
Determine chaperone # with PCR; run on agarose gel
  
−
e. 2x GoTaq Green
+
<b>New Chaperone PCR Mixture</b>
  
−
f. PCR Steps
+
- 2 uL overnight culture of bacterial clone (picked out of  amp+chlor)
  
−
g. Initial denaturation: 10 minutes at 94 degrees
+
- 0.4 uL primer cocktail with new chaperone primers equally mixed from 100 uM stocks
  
−
h. 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes
+
- 7.6 uL water
  
−
i. Final extension: 74 degrees, 5 minutes
+
- 10 uL 2x GoTaq Green
  
−
14. Determine origin with PCR; run on agarose gel
+
<b>New Chaperone PCR Thermal Profile</b>
  
−
a. Mixture
+
- Initial denaturation: 10 minutes at 94 degrees
  
−
b. 2 uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),  
+
- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes
  
−
c. 0.4 uL primer cocktail with origin primers 100 uM each
+
- Final extension: 74 degrees, 5 minutes
  
−
d. 7.6 uL water
+
<b>Origin PCR Mixture</b>
  
−
e. 10 uL 2x GoTaq Green
+
- 2 uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),
  
−
f. PCR Steps
+
- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks
  
−
g. Initial denaturation: 94 degrees, 10 minutes
+
- 7.5 uL water
  
−
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.
+
- 10 uL 2x GoTaq Green
  
−
i. Final extension: 74 degrees for 5 minutes
+
<b>Origin PCR Thermal Profile</b>
  
−
Plamids
+
- Initial denaturation: 94 degrees, 10 minutes
  
−
J119346
+
- 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
 +
 
 +
==Plasmids==
 +
 
 +
<b>J119346</b>
  
 
– High promoter
 
– High promoter
Line 91: Line 99:
 
– RFP
 
– RFP
  
−
J119347
+
<b>J119347</b>
  
 
– Low promoter
 
– Low promoter
Line 99: Line 107:
 
– GFP
 
– GFP
  
−
Origins
+
== Origins ==
 +
 
 +
<b>pSB1A2</b> High copy number
 +
 
 +
<b>J119310</b> Low copy number
  
−
pSB1A2 High copy number
+
==See also==
  
−
J119310 Low copy number
+
[[Repeating_20_Clone_Experiments|Repeating 20-clone experiments]]

Latest revision as of 18:37, 3 July 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 new chaperone primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Green

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

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

Plasmids

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

Origins

pSB1A2 High copy number

J119310 Low copy number

See also

Repeating 20-clone experiments

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