Preparation, storage, and use of 95% Formamide/1 mM EDTA in Infinium Assays

This article provides instructions for the proper preparation, storage, and use of the user-supplied reagent 95% formamide/1 mM EDTA in HD, HTS, and XT Infinium Assays. Background information on the reagent and its role in the assay workflow is also included.

A solution of 95% Formamide/1 mM EDTA (Ethylenediaminetetraacetic acid) is used during XStain, the single-base extension and staining step of the HD, HTS, and XT Infinium Assays. This reagent is user-supplied and proper preparation and storage are critical to obtaining optimal results.

Specifically, the 95% Formamide/1 mM EDTA solution is used to strip target DNA from the probe following single-base extension and prior to staining. This step is critical to ensure that staining reagents have unobstructed access to the newly incorporated single-base extension nucleotide.

Formamide acts as a denaturing agent by disrupting the hydrogen bonds between base pairs in double-stranded DNA. Once formamide is opened and exposed to oxygen, it begins to oxidize and convert to formic acid, which is less effective at denaturing DNA. For this reason, it is important to tightly seal the tubes after preparation, 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 in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety (EHS) information, refer to the applicable Safety Data Sheet (SDS), available through the Illumina Safety Data Sheets website.

Table 1. List of reagents and storage conditions.

Stock Consumable*	Supplier	Storage
EDTA, 0.5 M	Millipore, Catalog # 4056	Post-amp (-25°C to -15°C)
Sigma-Aldrich, catalog # E7889
Formamide, OmniPur	USA: VWR, catalog # EM-4650 EUR: catalog # CALB4610	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 aliquot into 15 ml or 50 ml conical tubes.

• Use conical, screw-top tubes rather than pop-top 1.5 ml tubes, as screw-cap tubes provide a tighter seal and better protection from oxidation.

• Label each tube with a use-by date of 6 months from the date of preparation.

• Store sealed tubes at -25°C to -15°C for up to 6 months and retrieve aliquots as needed.

• Use the solution on the same day the tube is opened and discard any unused volume

Table 2. 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

Suggested aliquot volumes

Manual workflow:

• Use 750 µl of 95% Formamide/1 mM EDTA per BeadChip. To allow sufficient volume and handling margin, prepare aliquots of at least 1 ml per BeadChip.

• Aliquot 1 ml of solution into 15 ml conical tubes.

Automated workflow (Tecan users):

• Aliquot into tube volumes appropriate for the laboratory workflow throughput.

• Discard any remaining volume if not used on the same day the tube is opened.

• Add the following volumes of 95% Formamide/1 mM EDTA to the reservoir based on batch size..

Table 3. Batch size and solution volume required.

Number of BeadChips	Reservoir Volume
1 - 8	15 ml
9 - 16	17 ml
17 - 24	25 ml

For additional information, refer to the Infinium Assay Lab Setup & Best Practices guide.

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