I've carried out some restriction enzyme digests, using a restriction enzyme StyI for Duffy Antigen gene and I get a ghost bands as well as my expected product sizes. Any ideas on how to go about this.
I tried reducing/increasing the PCR amplicon, increasing the time, reducing/increasing the concentration the restriction enzyme. Thank you and looking forward to your suggestion
can we see a picture please. I do not know what a ghost band is
thank you
I think I know what you are talking about. I have seen that some restriction enzymes cut more than they should if too much of them is added or if the reaction is left for too long. It might help to decrease the length or concentration of your reaction.
According to NEB troubleshooting guide. I have tried it, it worked out for me.
Extra bands in the gel If larger bands than expected are seen in the gel, this may indicate binding of the enzyme(s) to the substrate
- Lower the number of units in the reaction
- Add SDS (0.1–0.5%) to the loading buffer to dissociate the enzyme from the substrate
Star activity
- Use the recommended buffer supplied with the restriction enzyme
- Decrease the number of enzyme units in the reaction
- Make sure the amount of enzyme added does not exceed 10% of the total reaction volume. This ensures that the total glycerol concentration does not exceed 5% v/v
- Decrease the incubation time. Using the minimum reaction time required for complete digestion will help prevent star activity.
- Try using a High-Fidelity (HF) restriction enzyme. HF enzymes have been engineered for reduced star activity.
Partial restriction enzyme digest
- Enzymes that have low activity in salt-containing buffers (e.g., NEBuffer 3.1) may be salt sensitive. Make sure to clean up the DNA prior to digestion.
- DNA purification procedures that use spin columns can result in high salt levels, which inhibit enzyme activity. To prevent this, DNA solution should be no more than 25% of total reaction volume
- Clean-up the PCR fragment prior to restriction digest
- Use the recommended buffer supplied with the restriction enzyme
- Use at least 3–5 units of enzyme per μg of DNA
- Digest the DNA for 1–2 hours
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