Preparation, storage, and use of 95% formamide + EDTA in Infinium assays

Background Information 95% Formamide + 1 mM EDTA is used during XStain, the single-base extension, and staining portion of the Infinium assay. This solution is user-supplied and proper preparation and storage are critical to obtaining optimal results.

Specifically, 95% Formamide + 1 mM EDTA functions to strip the target DNA off of the probe after single-base extension and prior to staining. This is important for providing the staining reagents unencumbered access to the newly added single-base extension nucleotide.

Formamide functions as a denaturing agent by interfering with the hydrogen bonds between base pairs of double-stranded DNA. Once a bottle of formamide is opened and exposed to oxygen, it starts to oxidize and convert to formic acid, which is not as effective at denaturing DNA. For this reason, it is important to tightly seal tubes after preparation, always store the reagent frozen, and discard any unused portion of an aliquot after use.

WARNING: This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and a laboratory coat appropriate for the risk of exposure. Handle used reagents as chemical waste and discard them in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, see refer to the SDS, searchable on the page here.

Reagents

Stock Consumable*	Supplier	Storage
EDTA, 0.5 M	EMD Chemicals, catalog # 4056	Post-amp (-25°C to -15°C)
Sigma-Aldrich, catalog # E7889
Formamide, OmniPur	VWR, Int’l, catalog # EM-4650	Post-amp (-25°C to -15°C)
*Stock bottles must be replaced with new bottles every 12 months.

Preparation of Reagent

• Prepare large batches and divide them into 15 ml or 50 ml conical tubes.

• Use conical, screw-top tubes (as opposed to pop-top 1.5 ml tubes) because these have a tighter seal and offer better protection from oxidation.

• Label tubes with a ‘use-by’ date of 6 months from the date of preparation.

• Store sealed tubes for up to 6 months at -25°C to -15°C and use the stored mixture as needed.

Use the mixture on the same day you open the tube and discard any unused volume.

Stock Consumable*	Supplier	Storage
EDTA, 0.5 M	EMD Chemicals, catalog # 4056	Post-amp (-25°C to -15°C)
Sigma-Aldrich, catalog # E7889
Formamide, OmniPur	VWR, Int’l, catalog # EM-4650	Post-amp (-25°C to -15°C)
*Stock bottles must be replaced with new bottles every 12 months.

Suggested aliquot volumes Manual workflow:

• 750 µl of 95% Formamide + EDTA are used per beadchip, so aliquots of at least 1 ml are recommended for 1 aliquot per beadchip.

• Aliquot 1 ml volume into 15 ml conical tubes.

Automated workflow (Tecan users):

• Aliquot into tube volumes appropriate for the throughput of the lab workflow.

• Leftover volumes must be discarded if not used within the same day.

• The following volumes of 95% formamide + 1 mM EDTA are added to the reservoir:

Recipe for 95% Formamide + 1 mM EDTA
Desired Final Volume (ml)	100% Formamide stock (ml)	0.5 M EDTA stock (ml)	Water (ml)
10	9.50	0.02	0.48
15	14.25	0.03	0.72
20	19.00	0.04	0.96
25	23.75	0.05	1.20
50	47.50	0.10	2.40
100	95.00	0.20	4.80

See the Infinium Assay Lab Setup & Best Practices for additional information.

For any feedback or questions regarding this article (Illumina Knowledge Article #2232), contact Illumina Technical Support [e-mail].