Affymetrix DNA labelling for gene expression arrays: Difference between revisions
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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 === |
Revision as of 07:06, 26 January 2007
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. You could probably consider a gel shift assay.
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
- 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 ('''~~~~''') to the end of your tip.
References
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.