Validated GMO inactivation - Revision history

Rachel at 22:24, 15 December 2023

2023-12-15T22:24:11Z

Rachel at 07:51, 1 December 2023

2023-12-01T07:51:40Z

Rachel at 05:09, 30 November 2023

2023-11-30T05:09:58Z

Rachel at 17:42, 15 November 2023

2023-11-15T17:42:16Z

Rachel at 17:41, 15 November 2023

2023-11-15T17:41:19Z

Rachel at 17:28, 15 November 2023

2023-11-15T17:28:12Z

Rachel at 17:21, 15 November 2023

2023-11-15T17:21:34Z

Rachel at 17:18, 15 November 2023

2023-11-15T17:18:58Z

Rachel at 15:55, 15 November 2023

2023-11-15T15:55:57Z

Rachel at 15:33, 15 November 2023

2023-11-15T15:33:36Z

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 ([[OGMinact validation|versionFR]])
-In order to more formally validate the usual GMO inactivation done with bleach for disposal of plates and cultures, we are adding this page.  This validation was performed with commercial bleach (Coop 'Prix Garantie') and is applicable to both GMO bacteria and nematode worms transformed with antibioticR plasmids.  It is directly specific for [https://drive.google.com/file/d/1_db3H7Ybm_kd7yV-wnMKF55Snr805TAp/view?usp=sharing this SOP] of the lab collection.
+In order to more formally validate the usual GMO inactivation done with bleach for disposal of plates and cultures, we did more documented experiments that are being visually summarised in this page.  The validations were performed with commercial bleach (Coop 'Prix Garantie') and are applicable to both GMO bacteria and nematode worms transformed with antibioticR plasmids.  It is directly specific for [https://drive.google.com/file/d/1_db3H7Ybm_kd7yV-wnMKF55Snr805TAp/view?usp=sharing this SOP] of the lab collection.
 As always, precautions in using bleach and appropriate personal protective equipment (lab coat, gloves, and eye protection) are an essential part of this inactivation method.

← Older revision		Revision as of 07:51, 1 December 2023
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	A similar validation was performed with transformed ''C. elegans'' 'worm' cultures, to be put [[Worm validations\|on a new page]].		A similar validation was performed with transformed ''C. elegans'' 'worm' cultures, to be put [[Worm validations\|on a new page]].
		+
		+
		+	A [[Report ValidTestsGMOinact\|more formal report of the validation tests]] can also be read here.

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 ([[OGMinact validation|versionFR]])
-In order to more formally validate the usual GMO inactivation done with bleach for disposal of plates and cultures, we are adding this page.  This validation was performed with two brands of commercial bleach and is applicable to both GMO bacteria and nematode worms transformed with antibioticR plasmids.  It is directly specific for [https://drive.google.com/file/d/1_db3H7Ybm_kd7yV-wnMKF55Snr805TAp/view?usp=sharing this SOP] of the lab collection.
+In order to more formally validate the usual GMO inactivation done with bleach for disposal of plates and cultures, we are adding this page.  This validation was performed with commercial bleach (Coop 'Prix Garantie') and is applicable to both GMO bacteria and nematode worms transformed with antibioticR plasmids.  It is directly specific for [https://drive.google.com/file/d/1_db3H7Ybm_kd7yV-wnMKF55Snr805TAp/view?usp=sharing this SOP] of the lab collection.
 As always, precautions in using bleach and appropriate personal protective equipment (lab coat, gloves, and eye protection) are an essential part of this inactivation method.

@@ Line 58: / Line 58: @@
 No colonies were seen on these secondary plates exposed to bleach, even from the liquid recuperated from the plate treated with 1/10 bleach.
-We note in the experiment that we could also have tried to recuperate plasmids from the treated liquids, but that further test would only provide very useful information if very good competent cells were used. (sort of a waste)
+We note [https://www.evernote.com/shard/s586/sh/9e3be51e-434c-6b7d-25ed-ecb34b5bfe2a/HNkV4W3AW3UMJNqd7--KWVclJ5uwYYFC3Xq7oAL2MPJkgITpTeUvG5wErw in the experiment] that we could also have tried to recuperate plasmids from the treated liquids, but that further test would only provide very useful information if very good competent cells were used. (sort of a waste)
 No new colony growth even for the 1/10 treatment seemed quite satisfactory...

← Older revision		Revision as of 17:41, 15 November 2023
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−	The '''validation for bacterial plates''' was done upon bacteria transformed with a pJAMA-GFP plasmid, which make bright green colonies...	+	The '''validation for bacterial plates''' was done upon bacteria transformed with a pJAMA-GFP plasmid, which make bright green fluorescent colonies...

	A fresh 2ml culture was used to make three plates of cultures on growth media carrying the selective antibiotic, ampicillin (LB-amp100 plates). A glass spreader was used - from a bent pasteur pipette, to distribute the following liquids.		A fresh 2ml culture was used to make three plates of cultures on growth media carrying the selective antibiotic, ampicillin (LB-amp100 plates). A glass spreader was used - from a bent pasteur pipette, to distribute the following liquids.

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 These are shown below:
 [[File:Colonies for inactivation test.jpg|thumb|Plates used for bacterial colony validation|alt=|center]]
-[[File:Fluorbacts.jpg|center|thumb|fluorescence of pJAMA-GFP colonies for inactivation test]]
+[[File:Fluorbacts.jpg|center|thumb|Fluorescence colonies for the inactivation test]]
 Our 'DIY illuminator' with bright blue LEDs, and an amber filter (held in front of a phone camera) was used to get the fluorescence image.
 Then, the plates were treated with bleach in the big white tray.
-[[File:Tray inact.jpg|center|thumb|test of inactivation - bleach treatment]]Three conditions of inactivation for this validation experiment were run: full strength bleach, for plate 1, 1/10 bleach in water for plate 2, and water only for plate 3, with the least colonies.
+[[File:Tray inact.jpg|center|thumb|Test of inactivation - bleach treatment]]Three conditions of inactivation for this validation experiment were run: full strength bleach, for plate 1, 1/10 bleach in water for plate 2, and water only for plate 3, with the least colonies.
 ml of each solution was added to the appropriate plate, incubated for 5min and then swirled, as shown below, and incubated 5 more minutes.
-[[File:Swirling.jpg|center|thumb|swirling liquids on the plates after first incubations]]
+[[File:Swirling.jpg|center|thumb|Swirling liquids on the plates after first incubations.]]
 Then, after waiting 5 more minutes, a couple of mls of the liquid from each plate was recuperated into small conical tubes (15ml) with sterile serological pipets and the old Drummond pipet-aid.   On plate 1, all colonies appeared fully dissolved by the undiluted bleach, but plate 2 with 1/10 bleach, and plate 3, with only water, still had intact colonies, as shown.
 [[File:Plate1.jpg|center|thumb|After treatment, plate 1 colonies were dissolved away by the bleach.]]
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 No new colony growth even for the 1/10 treatment seemed quite satisfactory...
-[[File:Final inact.jpg|thumb|inactivation as usual, at the end]]
+[[File:Final inact.jpg|thumb|Inactivation as usual, at the end of the experiment.]]
 Finally, all secondary plates were inactivated as usual, according to the SOP, for proper disposal in the ordinary trash (one last picture here).
 A similar validation was performed with transformed ''C. elegans'' 'worm' cultures, to be put [[Worm validations|on a new page]].

← Older revision		Revision as of 17:21, 15 November 2023
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−	No colonies were seen on these secondary plates even from liquid recuperated from the plate treated with 1/10 bleach.	+
		+	No colonies were seen on these secondary plates exposed to bleach, even from the liquid recuperated from the plate treated with 1/10 bleach.

	We note in the experiment that we could also have tried to recuperate plasmids from the treated liquids, but that further test would only provide very useful information if very good competent cells were used. (sort of a waste)		We note in the experiment that we could also have tried to recuperate plasmids from the treated liquids, but that further test would only provide very useful information if very good competent cells were used. (sort of a waste)

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 [[File:Validated.jpg|center|thumb|All the secondary plates, after incubation overnight.]]
 Here is the fluorescence image (stacks of 3 plates).
-[[File:Fluorafter.jpg|center|thumb|fluorescent colonies were only found from the culture treated with water only.]]
+[[File:Fluorafter.jpg|center|thumb|Fluorescent colonies were only found from the culture treated with water only.]]

@@ Line 25: / Line 25: @@
 [[File:Fluorbacts.jpg|center|thumb|fluorescence of pJAMA-GFP colonies for inactivation test]]
+Our 'DIY illuminator' with bright blue LEDs, and an amber filter (held in front of a phone camera) was used to get the fluorescence image.
 Then, the plates were treated with bleach in the big white tray.
@@ Line 40: / Line 40: @@
-After all that, a repeat of the inactivation of all three initial plates, as per the SOP, with 100% javel put also on plate 2 and 3 was performed (esp as it was clear even the 1/10 solution is not dissolving the colonies, like the full strength one. The 'clean up' tray was carefully washed in back big sink, as usual (around in the intermediate Polymeka space).
 The 9 new plates were incubated overnight at 37<sup>o</sup>C and checked for growth.

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 ([[OGMinact validation|versionFR]])
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-A fresh 2ml culture was used to make three plates of cultures on growth media carrying the selective antibiotic, ampicillin (LB-amp100 plates).
+The '''validation for bacterial plates''' was done upon bacteria transformed with a pJAMA-GFP plasmid, which make bright green colonies...
 , with 100ul of culture (full lawn)
@@ Line 25: / Line 28: @@
 Then, the plates were treated with bleach in the big white tray.
-[[File:Tray inact.jpg|center|thumb|test of inactivation - bleach treatment]]
+[[File:Tray inact.jpg|center|thumb|test of inactivation - bleach treatment]]Three conditions of inactivation for this validation experiment were run: full strength bleach, for plate 1, 1/10 bleach in water for plate 2, and water only for plate 3, with the least colonies.