5-ROX, SE ([5-Carboxy-X-rhodamine, succinimidyl ester] *Single isomer*) is a labeling reagen used in the preparation of charge-modified dye-labeled Dideoxynucleotide Triphosphates (ddNTP) to "direct-load" DNA sequencing.

Instructions
I. Solution preparation
1. Storage solution preparation: 5-ROX, SE are dissolved in anhydrous DMSO or DMF. The concentration is generally recommended to be 1-10 mM. The specific concentration can be adjusted according to the experimental needs.
2. Working solution preparation: Use water or PBS to dilute the storage solution to a certain concentration. The specific concentration needs to be determined according to the experimental conditions.
II. Operation steps
1. Labeling reaction:
1) Mix the dissolved 5-ROX, SE with the amine-containing ddNTP solution.
2) The reaction is usually carried out at pH 8.0-9.0 to promote the reaction of NHS ester with the amine group in the ddNTP molecule.
3) The reaction time is usually 30 minutes to 1 hour. The reaction can be carried out at room temperature or overnight at 4°C. The specific reaction conditions are optimized according to the requirements of the experiment.
2. Removal of unreacted dye:
1) After the reaction is completed, use dialysis, gel filtration or centrifugation to remove the unreacted dye to avoid affecting the subsequent sequencing reaction.
2) These steps help ensure pure labeled ddNTPs for DNA sequencing.
3. DNA sequencing using labeled ddNTPs:
1) Use 5-ROX, SE labeled ddNTPs for DNA synthesis reactions. This method allows for direct loading of fluorescent labels during DNA synthesis for different base recognition.
2) The fluorescence properties of 5-ROX (Ex/Em: ~570/602 nm) allow it to be well distinguished from other commonly used fluorescent labels in sequencing, helping to identify different bases.
Notes:
1. Labeled ddNTPs should be stored at -20°C to avoid repeated freezing and thawing to ensure label stability.
2. Unused 5-ROX, SE solutions should be kept at -20°C and avoid exposure to strong light to avoid fluorescence decay.