Desalting

Desalting is a critical step in processing samples that have been isolated in the presence of salts and other small molecules. Resin-based desalting is performed by allowing the small molecules to freely enter the resin pore and be retarded in their flow through the packed resin bed while the high molecular weight molecules like proteins in the sample are excluded from the resin and rapidly exit the column. The exclusion limit of the resin is 7kD (Proteins)

The spin column and plate (96-well, and 384-well) method eliminates cumbersome column preparation or equilibration, allowing multiple-sample processing in less than 10 minutes. Samples containing as low as 20 μg per mL of proteins can be processed with ≥ 95% retention of salts and other small molecules (less than 1,000 Da)

Features:

Easy-to-use- no cumbersome column preparation or equilibration
Fast- no waiting for protein to emerge by gravity-flow
High protein recovery- low non-specific protein binding resin maximizes protein recovery
The sample volume is 20 μL to 4 mL

Protocol for Desalting or Removing Small Molecules

Equilibrate Coral 96-well Desalt Spin Plates to room temperature
Remove the sealing material from the bottom of the plate and place it on top of a wash plate.
Remove the sealing material from the top of the desalt plate.
Place the assembly into a centrifuge with 96-well plate-carrier rotor and centrifuged at 1,000×g for 2 minutes to remove the storage buffer. Discard the flow-through.
Rinse the wash plate three times with deionized water, dry and save for future use.
Stack the desalt plate on top of a sample collection plate (blue), aligning the alphanumeric indices on the plates.
Apply sample (20-100µl) to the center of the resin bed. To expel the entire sample, carefully touch pipette tip to the resin. For 20 µl protein samples (>300 µg/ml), apply a 20 µl stacker of water or buffer on top of resin bed after the sample wash has fully absorbed to ensure maximal protein recovery.
Centrifuge the plate assembly at 1,000×g for 2 minutes to collect the desalt sample. Discard the desalt plate or reserve it for further balancing purpose.

Coral Sephadex Packed Plates

Dye – Terminator removal is one of the major cleanup objective to yield a highly purified product prior to conducting a sequencing reaction. OrochemSephadex packed plates provide a very cost effective solution for high performance parallel processing of 96 sequencing reactions by gel filtration.

OrochemSephadex packed plates are available in 96-wells and 384-wells formats.
These plates can be used with gel filtration media for high throughput sequencing reaction cleanup.
They are constructed from rigid polystyrene that can withstand centrifugation.

General Protocol

Place the Sephadex plate over appropriate wash plates and place it into a centrifuge with correct carrier.
Centrifuge for 1 minute at 750 ×g to remove residual buffer. Discard the wash plates containing excess water.
Carefully and rapidly load 50 µl sequencing reaction mixture before the Spheadex gel dries out.
Centrifuge for 2 minutes at 750 ×g to eliminate big dye terminators, salts, and low molecular weight species.
Purified DNA of the sequencing reaction is recovered in collection plate