DIGscreen - Revision history

Xenbase at 15:05, 6 October 2023

2023-10-06T15:05:54Z

← Older revision		Revision as of 15:05, 6 October 2023
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	<span style="font-size:11.0pt;line-height:107%;font-family:		<span style="font-size:11.0pt;line-height:107%;font-family:
	"Calibri",sans-serif">[[Image:Digscreen_image1.~~JPG~~]]</span>		"Calibri",sans-serif">[[Image:Digscreen_image1.png]]</span>

	= 2. DIG high prime DNA label =		= 2. DIG high prime DNA label =
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	<span style="font-size:11.0pt;line-height:107%;font-family:		<span style="font-size:11.0pt;line-height:107%;font-family:
	"Calibri",sans-serif">[[Image:DIGscreen image2.~~jpg~~\|410x262px]]</span>		"Calibri",sans-serif">[[Image:DIGscreen image2.png\|410x262px]]</span>

	Compare the intensity of the spots out of your labeling reaction to the control and calculate the amount of DIG-labeled DNA. If the 0.1 pg dilution spots of your probe and of the control are visible, then the labeled probe has reached the expected labeling efficiency. ''''''		Compare the intensity of the spots out of your labeling reaction to the control and calculate the amount of DIG-labeled DNA. If the 0.1 pg dilution spots of your probe and of the control are visible, then the labeled probe has reached the expected labeling efficiency. ''''''
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	<span style="line-height:111%">16)<span style="font:7.0pt "Times New Roman""> </span></span>Expose 30min & develop film.		<span style="line-height:111%">16)<span style="font:7.0pt "Times New Roman""> </span></span>Expose 30min & develop film.

	[[Image:DIGscreen image3.~~jpg~~]]<span style="font-size:11.0pt;line-height:		[[Image:DIGscreen image3.png]]<span style="font-size:11.0pt;line-height:
	107%"> </span>		107%"> </span>

Ctelmer: /* 3. Determination of labeling efficiency-Dilution and dot blot */

2023-10-04T19:37:19Z

3. Determination of labeling efficiency-Dilution and dot blot

← Older revision		Revision as of 19:37, 4 October 2023
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	(For reagent and more details, please refer to ~~''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at~~ [~~http~~://www.~~genomex~~.com/~~Downloads~~/~~Sequencing_Site.pdf <span style="color:blue">http:~~//~~www.genomex.com~~/~~Downloads/Sequencing_Site.pdf</span>~~]~~[http://www.genomex.com/Downloads/Sequencing_Site~~.~~pdf <span style="color:black;text-decoration:none">~~)~~</span>]~~ *All ~~solution comes~~ from the Kit.		(For reagent and more details, please refer to [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All solutions come from the Kit.

Ctelmer: /* 2. DIG high prime DNA label */

2023-10-04T19:36:08Z

2. DIG high prime DNA label

← Older revision		Revision as of 19:36, 4 October 2023
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	DNA is random primed labeled with Digoxigenin-11-dUTP using DIG-High Prime, a ~~5 ×~~ concentrated labeling mixture of random hexamers, dNTP mix containing alkali-labile		DNA is random primed labeled with Digoxigenin-11-dUTP using DIG-High Prime, a 5X concentrated labeling mixture of random hexamers, dNTP mix containing alkali-labile

	Digoxigenin-11-dUTP, labeling grade Klenow enzyme and an optimized reaction buffer.		Digoxigenin-11-dUTP, labeling grade Klenow enzyme and an optimized reaction buffer.

Ctelmer: /* 4. Screen library */

2023-10-04T19:34:56Z

4. Screen library

Ctelmer: /* 4. Screen library */

2023-10-04T19:31:42Z

4. Screen library

← Older revision		Revision as of 19:31, 4 October 2023
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	''' '''		''' '''

	(For reagent and more details, please refer to [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All ~~solution comes~~ from the Kit.		(For reagent and more details, please refer to [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All solutions come from the Kit.

Ctelmer: /* 5. Immunological Detection */

2023-10-04T19:31:19Z

5. Immunological Detection

← Older revision		Revision as of 19:31, 4 October 2023
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	= 5. Immunological Detection =		= 5. Immunological Detection =

	(Please refer to protocol of ~~''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at~~		(Please refer to protocol of [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All solutions come from the Kit.

	[~~http~~://www.~~genomex~~.com/~~Downloads~~/~~Sequencing_Site.pdf <span style="color:blue">http:~~//~~www.genomex.com~~/~~Downloads/Sequencing_Site.pdf</span>~~]~~[http://www.genomex~~.~~com/Downloads/Sequencing_Site.pdf <span style="color:black;text-decoration:none">~~)~~</span>]~~ *All ~~solution comes~~ from the Kit.

Ctelmer: /* 4. Screen library */

2023-10-04T19:29:43Z

4. Screen library

← Older revision		Revision as of 19:29, 4 October 2023
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	''' '''		''' '''

	(For reagent and more details, please refer to ~~''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at~~ [~~http~~://www.~~genomex~~.com/~~Downloads~~/~~Sequencing_Site.pdf <span style="color:blue">http:~~//~~www.genomex.com~~/~~Downloads/Sequencing_Site.pdf</span>~~]~~[http://www.genomex.com/Downloads/Sequencing_Site~~.~~pdf <span style="color:black;text-decoration:none">~~)~~</span>]~~ *All solution comes from the Kit.		(For reagent and more details, please refer to [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All solution comes from the Kit.

Ctelmer: /* 2. DIG high prime DNA label */

2023-10-04T19:28:19Z

2. DIG high prime DNA label

← Older revision		Revision as of 19:28, 4 October 2023
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	(For reagent and more details, please refer to ~~''DIG-High Prime DNA Labeling and Detection Starter'' Kit I at~~ [~~http~~://www.~~genomex~~.com/~~Downloads~~/~~Sequencing_Site.pdf <span style="color:blue">http:~~//~~www.genomex.com~~/~~Downloads/Sequencing_Site.pdf</span>~~]~~[http://www.genomex.com/Downloads/Sequencing_Site~~.~~pdf <span style="color:black;text-decoration:none">~~)~~</span>]~~ *All ~~solution comes~~ from the Kit.		(For reagent and more details, please refer to [https://www.sigmaaldrich.com/US/en/product/roche/11745832910?gclid=Cj0KCQjwmvSoBhDOARIsAK6aV7g-Kc5DDj0UgH_5dn46wW_dMzd_uYX8rZ8nib91rY_1Ad9eeElC9gkaAuzfEALw_wcB DIG-High Prime DNA Labeling and Detection Starter Kit I].) *All solutions come from the Kit.

Ctelmer: /* 1. PCR DIG Labeling */

2023-10-04T19:24:41Z

1. PCR DIG Labeling

← Older revision		Revision as of 19:24, 4 October 2023
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	<span style="line-height:111%">6)<span style="font:7.0pt "Times New Roman""> </span></span>For PCR product, clean up with ~~<span style="color~~:~~red">zoymo~~ DNA ~~clean</span>~~. Check OD260.		<span style="line-height:111%">6)<span style="font:7.0pt "Times New Roman""> </span></span>For PCR product, clean up with [https://www.zymoresearch.com/collections/dcc-pcr-purification-kits Zymo DNA Clean]. Check OD260.

Ctelmer: /* 1. PCR DIG Labeling */

2023-10-04T19:17:38Z

1. PCR DIG Labeling

← Older revision		Revision as of 19:17, 4 October 2023
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	This is the standard method, for alternative labeling method, please see #2.		This is the standard method, for alternative labeling method, please see #2.

	(For reagent and more details, please refer to kit protocol ~~at –~~		(For reagent and more details, please refer to the [https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/287/736/dig-application-manual-for-nonradioactive-in-situ-hybridisation-iris.pdf kit protocol].)

	[http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm <span style="color:purple">http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm</span>][http://www.roche-applied-science.com/PROD_INF/MANUALS/DIG_MAN/dig_toc.htm <span style="color:black;text-decoration:none">)</span>] For each experimental or control sample, add the following components to a sterile microcentrifuge tube. Place the tube on ice during pipetting.		For each experimental or control sample, add the following components to a sterile microcentrifuge tube. Place the tube on ice during pipetting.

	Steps:		Steps:

← Older revision		Revision as of 19:34, 4 October 2023
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	1) Stripping filters		1) Stripping filters

	'''''(Important Tip: Membranes should never be allowed to dry before stripping.''''' ''Once dried, the membrane cannot be stripped and reprobed.)''		('''''Important Tip: Membranes should never be allowed to dry before stripping.''''' ''Once dried, the membrane cannot be stripped and reprobed.'')

	Stripping DIG-labeled DNA Probe after Chemiluminescent Detection		Stripping DIG-labeled DNA Probe after Chemiluminescent Detection

	<span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane thoroughly with ~~bouble~~ distilled water for 1 min		<span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane thoroughly with double distilled water for 1 min

	<span style="line-height:150%">•<span style="font:7.0pt "Times New Roman""> </span></span>Wash membrane twice at 37°C in Stripping Buffer (0.2 M NaOH containing 0.1% SDS) for 15 min each.		<span style="line-height:150%">•<span style="font:7.0pt "Times New Roman""> </span></span>Wash membrane twice at 37°C in Stripping Buffer (0.2 M NaOH containing 0.1% SDS) for 15 min each.

	<span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane with 2× SSC for 5 min		<span style="line-height:111%">•<span style="font:7.0pt "Times New Roman""> </span></span>Rinse membrane with 2X SSC for 5 min



	<span style="line-height:111%">2)<span style="font:7.0pt "Times New Roman""> </span></span>~~Prehybrization~~ in 40ml DIG Easy Hyb at 48°C for 1 hour		<span style="line-height:111%">2)<span style="font:7.0pt "Times New Roman""> </span></span>Prehybridization in 40ml DIG Easy Hyb at 48°C for 1 hour

	<span style="line-height:149%">3)<span style="font:7.0pt "Times New Roman""> </span></span>Denature the probe--all probes mixed in 350ul 10uM Tris and denatured under 95°C for 5min, then chill quickly on ice bath		<span style="line-height:149%">3)<span style="font:7.0pt "Times New Roman""> </span></span>Denature the probe--all probes mixed in 350ul 10uM Tris and denatured under 95°C for 5min, then chill quickly on ice bath

	<span style="line-height:149%">4)<span style="font:7.0pt "Times New Roman""> </span></span>~~Hybrization~~ solution-- Immediately add the denatured probe to a tube containing the appropriate amount of prewarmed DIG Easy Hyb (3.5 ml per 100 cm2) and		<span style="line-height:149%">4)<span style="font:7.0pt "Times New Roman""> </span></span>Hybridization solution-- Immediately add the denatured probe to a tube containing the appropriate amount of prewarmed DIG Easy Hyb (3.5 ml per 100 cm2) and

	mix by inversion (avoid foaming) to form the hybridization solution		mix by inversion (avoid foaming) to form the hybridization solution

	<span style="line-height:154%">5)<span style="font:7.0pt "Times New Roman""> </span></span>Pour off the prehybridization buffer.<span style="color:#F46E20"> </span>'''Immediately''' add hybridization solution containing DIG-labeled probe and ~~hybrization~~ at 48°C for 6-16 hours		<span style="line-height:154%">5)<span style="font:7.0pt "Times New Roman""> </span></span>Pour off the prehybridization buffer.<span style="color:#F46E20"> </span>'''Immediately''' add hybridization solution containing DIG-labeled probe and hybridization at 48°C for 6-16 hours

	<span style="line-height:150%">6)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with low Stringency Buffer (1L ~~2XSSC~~ containing 0.1% SDS) at room temperature for 15min twice		<span style="line-height:150%">6)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with low Stringency Buffer (1L 2X SSC containing 0.1% SDS) at room temperature for 15min twice

	<span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman""> </span></span>Preheat high Stringency Buffer (1L 0.~~5XSSC~~ containing 0.1% SDS) into 65°C		<span style="line-height:111%">7)<span style="font:7.0pt "Times New Roman""> </span></span>Preheat high Stringency Buffer (1L 0.5X SSC containing 0.1% SDS) into 65°C

	<span style="line-height:149%">8)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with preheated high Stringency Buffer (1L 0.~~5XSSC~~ containing 0.1% SDS) at 65°C for 15min twice		<span style="line-height:149%">8)<span style="font:7.0pt "Times New Roman""> </span></span>Wash with preheated high Stringency Buffer (1L 0.5X SSC containing 0.1% SDS) at 65°C for 15min twice

	= 5. Immunological Detection =		= 5. Immunological Detection =