Affymetrix DNA labelling for gene expression arrays - Revision history

Torsten Waldminghaus at 17:31, 23 February 2009

2009-02-23T17:31:00Z

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	== Affymetrix DNA labelling for gene expression arrays ==		== Affymetrix DNA labelling for gene expression arrays ==

Reshma P. Shetty at 01:29, 9 May 2007

2007-05-09T01:29:29Z

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	*mail to jamesdothadfieldatcancerdotorgdotuk		*mail to jamesdothadfieldatcancerdotorgdotuk
	*This page was created by James Hadfield on 17 October 2006. It was based on a protocol I recieved from Justin Borevitz 3/19/2004, hope you found it useful.		*This page was created by James Hadfield on 17 October 2006. It was based on a protocol I recieved from Justin Borevitz 3/19/2004, hope you found it useful.
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Jamesh008: /* Labeling QC */

2007-01-26T14:14:30Z

Labeling QC

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	Use 5ul to check yield and quality on a gel (Figure 1).		Use 5ul to check yield and quality on a gel (Figure 1).

-	[[Image:bioprime_labeled_DNA.jpg\|thumb\|200px\|~~left~~\|BioPrime labeled DNA.]]	+	[[Image:bioprime_labeled_DNA.jpg\|thumb\|200px\|right\|BioPrime labeled DNA.]]

Jamesh008: /* Labeling QC */

2007-01-26T14:14:07Z

Labeling QC

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	All bands should be close to the same intensity.		All bands should be close to the same intensity.
-	~~You could probably consider a gel shift assay.~~

	=== Hybridisation ===		=== Hybridisation ===

Jamesh008: /* Labeling QC */

2007-01-26T14:06:59Z

Labeling QC

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	[[Image:bioprime_labeled_DNA.jpg\|thumb\|200px\|left\|BioPrime labeled DNA.]]		[[Image:bioprime_labeled_DNA.jpg\|thumb\|200px\|left\|BioPrime labeled DNA.]]
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	All bands should be close to the same intensity.		All bands should be close to the same intensity.
		+	You could probably consider a gel shift assay.

	=== Hybridisation ===		=== Hybridisation ===

Jamesh008: /* Labeling QC */

2007-01-26T14:05:50Z

Labeling QC

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	Use 5ul to check yield and quality on a gel (Figure 1).		Use 5ul to check yield and quality on a gel (Figure 1).

		+	[[Image:bioprime_labeled_DNA.jpg\|thumb\|200px\|left\|BioPrime labeled DNA.]]
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	Figure 1. 5ul of Bioprime genome DNA labeling. Thanks Todd Michael.		Figure 1. 5ul of Bioprime genome DNA labeling. Thanks Todd Michael.

Jamesh008 at 08:34, 17 October 2006

2006-10-17T08:34:48Z

New page

== Affymetrix DNA labelling for gene expression arrays ==

This protocol will allow you to perform the labelling of DNA ready for Affymetrix expression genechip analysis.

==Workflow==

==Materials==
*BioPrime Random Genomic DNA Labeling kit
Do not store enzymes in a frost-free freezer.

Miscellaneous Reagents

==Protocol==

=== BioPrime Random Genomic DNA Labeling Protocol ===
Extract DNA from a single leaf of each of 4 Columbia and 4 Ler plants using Qiagen DNA easy plant prep kit and re-suspended in 100ul. To extract equal amount of DNA from the 50 – 100 mutant or wild type F2 plants, take 1 leaf (or equal amount of tissue) from each plant and freeze together in liquid nitrogen. Grind tissue together and use ~100mg powder in the Qiagen DNA easy kit. Resuspend DNA in 100ul.

Total labeling reaction volume is 150ul

add
~30ul (300ng) Plant DNA
60ul 2.5X random primers (Bioprime kit).
to 132ul with H20 (~42ul)

Denature at > 95C for 5-10minutes, then cool on ice.

add
15ul 10X dNTPs mix with Biotin dCTP
3ul Klenow polymerase enzyme

Incubate overnight @ 25C (room temp). This results in small labeled products about 50bp (Figure 1).

add
15ul 3M NaOAc
400ul cold 100% EtOH
spin, remove supernaunt, and wash with
500ul cold 75% EtOH
resuspend in
100ul water

=== Labeling QC ===
Use 5ul to check yield and quality on a gel (Figure 1).

Figure 1. 5ul of Bioprime genome DNA labeling. Thanks Todd Michael.

All bands should be close to the same intensity.

=== Hybridisation ===
The labeled DNA is then treated the same as labeled cRNA in standard hybridization protocols for gene expression, see Eukaryotic Target Preparation Pg 51 [https://www.affymetrix.com/Download/manuals/expression_ever_manual.pdf] Briefly.

95ul Labeling reaction
3.3ul Control Oligo B2 (3nM)
10 ul Eukaryotic Hybridization Controls (optional)
2ul Herring Sperm DNA (10mg/ml)
2ul Acetylated BSA (50mg/ml)
110ul 2X hybridization buffer

Final Volume ~225ul Heat Denature >95C for 5 minutes, then cool Replace 1X hyb solution in pre wetted warmed chips. with 200ul (of ~225ul) labeled hybridization cocktail. Hybridize overnight @ 45C with rotation 60rpm. Wash and double stain with antibody.

==Notes==
#List troubleshooting tips here.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding <font face="courier"><nowiki>('''~~~~''')</nowiki></font> to the end of your tip.

==References==

<biblio>
# none
</biblio>

==Contact==
*mail to jamesdothadfieldatcancerdotorgdotuk
*This page was created by James Hadfield on 17 October 2006. It was based on a protocol I recieved from Justin Borevitz 3/19/2004, hope you found it useful.