Affymetrix DNA labelling for gene expression arrays

Affymetrix DNA labelling for gene expression arrays

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

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).

BioPrime labeled DNA.

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 [1] 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

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

Please sign your name to your note by adding ('''~~~~''') to the end of your tip.

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.