Eckdahl-replication-protocol - Revision history

Jolim at 18:37, 3 July 2014

2014-07-03T18:37:30Z

Jolim: Added "See also" section

2014-07-03T18:36:23Z

Added "See also" section

ToEckdahl at 16:31, 4 June 2014

2014-06-04T16:31:23Z

ToEckdahl at 16:28, 4 June 2014

2014-06-04T16:28:25Z

ToEckdahl at 16:27, 4 June 2014

2014-06-04T16:27:32Z

ToEckdahl at 16:26, 4 June 2014

2014-06-04T16:26:50Z

ToEckdahl at 16:17, 4 June 2014

2014-06-04T16:17:40Z

Jolim at 18:01, 21 May 2014

2014-05-21T18:01:09Z

Jolim: 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..."

2014-05-21T15:03:57Z

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..."

New page

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

@@ Line 88: / Line 88: @@
 - Final extension: 74 degrees for 5 minutes
 ==Plasmids==
@@ Line 116: / Line 112: @@
 	<b>J119310</b> Low copy number

← Older revision		Revision as of 18:36, 3 July 2014
Line 88:		Line 88:

	- Final extension: 74 degrees for 5 minutes		- Final extension: 74 degrees for 5 minutes
		+
		+	==See also==
		+
		+	[[Repeating_20_Clone_Experiments\|Repeating 20-clone experiments]]

	==Plasmids==		==Plasmids==

@@ Line 89: / Line 89: @@
 - Final extension: 74 degrees for 5 minutes
-==Plamids==
+==Plasmids==
 <b>J119346</b>

@@ Line 55: / Line 55: @@
 - 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
+- 0.4 uL primer cocktail with new chaperone primers equally mixed from 100 uM stocks
 - 7.6 uL water

@@ Line 59: / Line 59: @@
 - 7.6 uL water
-- 10 uL 2x GoTaq Gree
+- 10 uL 2x GoTaq Green
 <b>New Chaperone PCR Thermal Profile</b>
@@ Line 68: / Line 68: @@
 - Final extension: 74 degrees, 5 minutes
 <b>Origin PCR Mixture</b>

@@ Line 51: / Line 51: @@
 Determine chaperone # with PCR; run on agarose gel
-<b>Mixture</b>
+<b>New Chaperone PCR Mixture</b>
-uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),
+- 2 uL overnight culture of bacterial clone (picked out of  amp+chlor)
-water
+- 0.4 uL primer cocktail with origin primers equally mixed from 100 uM stocks
-x GoTaq Green
+- 7.6 uL water
-<b>PCR Steps</b>
+- 10 uL 2x GoTaq Gree
 - Initial denaturation: 10 minutes at 94 degrees
-- 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes
+- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes
 - Final extension: 74 degrees, 5 minutes
@@ Line 70: / Line 71: @@
 Determine origin with PCR; run on agarose gel
-<b>Mixture</b>
+<b>Origin PCR 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
+- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks
-- 7.6 uL water
+- 7.5 uL water
 - 10 uL 2x GoTaq Green
-<b>PCR Steps</b>
+<b>Origin PCR Thermal Profile</b>
 - Initial denaturation: 94 degrees, 10 minutes

@@ Line 84: / Line 84: @@
 - 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 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

@@ Line 1: / Line 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
-.	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
-.	Combine 0.5 mL of each clone.
+Prepare stock of .25 g/mL L-arabinose
-.	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
-.	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)
-.	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.
-.	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
-.	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
-.	Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.
+- 37degrees C overnight
-.	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)
-.	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.
-.	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.
-.	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>Mixture</b>
-f.	PCR Steps
+uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),
-g.	Initial denaturation: 10 minutes at 94 degrees
+water
-h.	30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes
+x GoTaq Green
-i.	Final extension: 74 degrees, 5 minutes
+<b>PCR Steps</b>
-.	Determine origin with PCR; run on agarose gel
+- Initial denaturation: 10 minutes at 94 degrees
-a.	Mixture
+- 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes
-b.	2 uL overnight culture of bacterial clone (picked out of  either amp or amp+chlor, according to the presence of the chaperone),
+- Final extension: 74 degrees, 5 minutes
-c.	0.4 uL primer cocktail with origin primers 100 uM each
+Determine origin with PCR; run on agarose gel
-d.	7.6 uL water
+<b>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
+- 0.4 uL primer cocktail with origin primers 100 uM each
-g.	Initial denaturation: 94 degrees, 10 minutes
+- 7.6 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>PCR Steps</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.
 –	High promoter
@@ Line 91: / Line 98: @@
 –	RFP
-	J119347
+<b>J119347</b>
 –	Low promoter
@@ Line 99: / Line 106: @@
 –	GFP
-Origins
+== Origins ==
-	pSB1A2 High copy number
+	<b>pSB1A2</b> High copy number
-	J119310 Low copy number
+	<b>J119310</b> Low copy number