Can I store the assembled products?
Yes, you can store them at 4℃ for a few days. For further long-term storage, add final 5 mM EDTA before storage at -20℃. The following OriCiro Amp Kit tolerates up to final 0.5 mM EDTA.
Do I need purification of the assembly products before the OriCiro amplification?
Can I use DNA fragments digested by restriction enzyme or CRISPR-Cas9?
No assembled products is observed
In certain conditions, the assembled product quantity may not be large enough to be observed after assembly reaction. It is highly recommended to perform following amplification reaction
Can I store the amplified products?
How can I analyze the amplified products by electrophoresis?
Dilute the 0.5 µl of products with 5 µl of Loading Buffer (25 mM Tris-HCl pH8.0, 25 mM EDTA, 0.1% SDS, 5% glycerol, 0.1% bromophenol blue), followed by agarose gel electrophoresis. The supercoiled products will be observed.
Extra bands are observed in agarose gel electrophoresis.
In case of DNA contamination.
Put negative control sample (reaction without DNA) for the amplification reaction. OriCiro Amp Kit amplifies DNA even from a single contamination molecule. To identify and remove DNA contamination, monoclonal amplification from a single DNA molecule following limiting dilution is useful.
In case of Concatemer DNA
When the amplified DNA size is less than 4 kbp, extra bands by concatemer DNA may appear. The situation may improve by shortening the reaction time.
No amplified products is observed
Please check the DNA size is less than 100 kbp. If the target DNA size exceeds 100 kbp, the amplification requires a different protocol than described in this manual.
Other possibility is the assembly reaction will be inhibited when salt and/or EDTA concentration of the DNA sample is too high.
- Increase the amplification reaction temperature up to 37℃.
- Increase the amplification reaction time up to 18 hours.
- Increase the template DNA amount added into the amplification reaction.
- Pre-incubate the amplification reaction mixture at 30℃ for 15 minutes just before adding the template DNA.
Can I reamplify the amplified products with OriCiro Amp Kit?
Dilute the amplified products (~10-6), prepare new amplification reaction mixture and incubate again. We checked the amplified products can reamplify continuously more than 10 times.