SYBR Green I DNA凝膠染色
SYBR Green I is fluorescent dye that binds specifically to double-stranded DNA. There are three variants of staining protocol: gel soaking, gel pre-staining, and sample pre-staining.
Gel soaking
Classical method for agarose and polyacrylamide gels.
- Run sample(s) in agarose or polyacrylamide gel.
- In a beaker, add 10 uL of 10,000x SYBR Green I solution in DMSO to 100 mL of 1x TE, TBE, or TAE buffer (for mini gel), or 50 uL of 10,000x SYBR Green I solution in DMSO to 500 mL of 1x TE, TBE, or TAE buffer (for mid-sized gel). Mix thoroughly with spatula, rod, or magnetic stirrer.
- Pour the diluted SYBR Green I solution in appropriate tray or pan.
- Soak the gel for 5-10 min.
- View or document the gel using 254 nm low-pressure mercury lamp and orange filter.
Gel pre-staining
This method is acceptable for agarose gels only, but not for PAAG.
- Boil the agarose in buffer to dissolution using microwave or heating appliance.
- While hot, add 1 uL of 10,000x SYBR Green I solution in DMSO per each 10 mL of gel solution. Mix thoroughly.
- Pour the gel and let it cool down.
- For best results, add 1 uL of of 10,000x SYBR Green I solution in DMSO per each 10 mL of buffer near anode (“+”, red wire).
- Run the samples. Real-time monitoring of migrating bands under 254 nm low-pressure mercury lamp possible.
- View or document the gel using 254 nm low-pressure mercury lamp and orange filter.
Sample pre-staining
Least sensitive, most economical method.
- Mix 25 uL of DMSO and 1 uL of 10,000x SYBR Green I solution in DMSO.
- Add 1 uL of the solution to each sample to be separated on agarose or polyacrylamide gel.
- Run the samples. Real-time monitoring of migrating bands under 254 nm low-pressure mercury lamp possible.
- View or document the gel using 254 nm low-pressure mercury lamp and orange filter.