AGiR! for genomic integrity/cometassaydev - Revision history

Rachel: /* Current 'best' protocol */

2021-01-16T11:27:11Z

Current 'best' protocol

Rachel at 09:22, 17 November 2019

2019-11-17T09:22:23Z

Rachel at 09:19, 17 November 2019

2019-11-17T09:19:21Z

Rachel at 14:08, 15 November 2019

2019-11-15T14:08:29Z

Rachel: /* Ideas for protocol improvement */

2019-11-13T16:54:49Z

Ideas for protocol improvement

Rachel: /* Ideas for protocol improvement */

2019-11-13T16:54:23Z

Ideas for protocol improvement

Rachel: /* Ideas for protocol improvement */

2019-11-13T16:52:15Z

Ideas for protocol improvement

Rachel: /* Ideas for protocol improvement */

2019-11-13T16:51:20Z

Ideas for protocol improvement

Rachel: /* Ideas for protocol improvement */

2019-11-13T16:49:26Z

Ideas for protocol improvement

Rachel: /* Ideas for 'Comet Chip' development as an 'automated' system */

2018-09-18T21:02:09Z

Ideas for 'Comet Chip' development as an 'automated' system

@@ Line 47: / Line 47: @@
 RT incubation (watch under scope to see cells 'disappearing' leaving nucleoids - usually only about 20 minutes max)<br>
 ''Both Detergent and PK are essential for this to occur without induction of apoptosis, which would ruin any hope for successful observation of comets.'' <br>
 ) as treatment seems sufficient, apply 1X TBE pH9 (alkaline treatment) to cell patches<br>

@@ Line 1: / Line 1: @@
-This is the default page for the concrete aspects of the cheek cell comet assay protocol. <br>
+This page describes some concrete aspects of the cheek cell comet assay protocol. <br>
 [[File:CheekCellComet 2dec16slide2x10GFPb.jpg|thumb|upright=0.9|Illustration of a "comet", the migration of the DNA after an electrophoresis]]
@@ Line 63: / Line 62: @@
 For running the comet assay, leaving the treatment overnight (PK buffer etc) has given very good results (in particular with Violaine, Vithoo and Jennifer in the summer of 2018).  They also showed clearly that the lab water used is critically important.  Just using distilled water from the shop has the potential to really increase apoptosis in the agarose patches, leading to 'no comet' results. <br>
 Additionally, so the patches stay flat during the run, it is a good idea to place the patches between 2 slides, as also successfully demonstrated in the summer of 2018. <br>
-Ultimately, the 'cheek cell chip' is hoped to make everything easy, to avoid having to do so many manipulations (even with paint-brushes). <br>
+Here are pages they put up about their work:<br>
 While we had tried to assess alternatives that would form a nicely colored DNA 'comet tail' for easier DIY image acquisition/analyses, but neither Hemalum after a MeOH:acetic acid (3:1) fix nor methylene blue aqueous seem very useful at all in this regard...  Some tests with methylene blue and iodine have been a bit promising, but further tests were generally not encouraging.  New ideas are very welcome!<br>
 <br>
@@ Line 75: / Line 77: @@
 <br>
-== Ideas for 'Comet Chip' development as an 'automated' system ==
+== Ideas for 'Cheek Cell Chip' development as an 'automated' system ==
 The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells.  In the context of the OpenFlexure scope, we may simply modify the upper stage attachment, to become the 'cheek cell chip' in fact.  But first we need to see more than just nuclei with this microscope/RaspberryPi system (work in progress!)!

← Older revision		Revision as of 09:19, 17 November 2019
Line 76:		Line 76:

	== Ideas for 'Comet Chip' development as an 'automated' system ==		== Ideas for 'Comet Chip' development as an 'automated' system ==
−	The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells. In the context of the OpenFlexure scope, we may simply modify the upper stage attachment, to become the 'cheek cell chip' in fact. But first we need to see more than nuclei!	+	The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells. In the context of the OpenFlexure scope, we may simply modify the upper stage attachment, to become the 'cheek cell chip' in fact. But first we need to see more than just nuclei with this microscope/RaspberryPi system (work in progress!)!

@@ Line 66: / Line 66: @@
 While we had tried to assess alternatives that would form a nicely colored DNA 'comet tail' for easier DIY image acquisition/analyses, but neither Hemalum after a MeOH:acetic acid (3:1) fix nor methylene blue aqueous seem very useful at all in this regard...  Some tests with methylene blue and iodine have been a bit promising, but further tests were generally not encouraging.  New ideas are very welcome!<br>
 <br>
-In the meantime, DIYmicroscopes that can use a blue LED excitation light are being worked upon, too!!  The [https://openflexure.org/ OpenFlexure] system for epifluor may just do the trick, we hope.  Perhaps we will modify the upper stage attachment, to become the 'cheek cell chip' in fact. A second build to their v6 still needs to be put together with the motors, and then we may really be golden, fingers crossed.<br>
+In the meantime, DIYmicroscopes that can use a blue LED excitation light are being worked upon, too!!  The [https://openflexure.org/ OpenFlexure] system for epifluor may just do the trick, we hope.
 <br>
 If you have trouble removing the coverslip from the patch of embedded cells without messing it up, you can add buffer first, or lose patches during manipulations, you can make an initial patch on the slide before adding the cell suspension in agarose. <br>
@@ Line 75: / Line 76: @@
 == Ideas for 'Comet Chip' development as an 'automated' system ==
-The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells.
+The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells.  In the context of the OpenFlexure scope, we may simply modify the upper stage attachment, to become the 'cheek cell chip' in fact.  But first we need to see more than nuclei!

@@ Line 61: / Line 61: @@
 ==Ideas for protocol improvement==
-For running the comet assay, leaving the treatment overnight (PK buffer etc) has given very good results (in particular with Violaine, Vithoo and Jennifer in the summer of 2018).  They also showed clearly that the lab water used is also very important.  Just using distilled water from the shop has the potential to really increase apoptosis in the agarose patches, leading to 'no comet' results. <br>
+For running the comet assay, leaving the treatment overnight (PK buffer etc) has given very good results (in particular with Violaine, Vithoo and Jennifer in the summer of 2018).  They also showed clearly that the lab water used is critically important.  Just using distilled water from the shop has the potential to really increase apoptosis in the agarose patches, leading to 'no comet' results. <br>
 Additionally, so the patches stay flat during the run, it is a good idea to place the patches between 2 slides, as also successfully demonstrated in the summer of 2018. <br>
 Ultimately, the 'cheek cell chip' is hoped to make everything easy, to avoid having to do so many manipulations (even with paint-brushes). <br>

← Older revision		Revision as of 21:02, 18 September 2018
Line 72:		Line 72:

	== Ideas for 'Comet Chip' development as an 'automated' system ==		== Ideas for 'Comet Chip' development as an 'automated' system ==
−	The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet ~~cells test~~ should be carried out on the chip, thus contain electrodes, etc...	+	The idea is to make an open-source microfluidic device or something inspired by microfluidics. The chip should be easy to make and to use. All the comet cell treatments should be carried out on the chip, thus contain electrodes, etc... The dream configuration would allow micronuclei assays to be done first, and comets later on the same embedded cells.