Oct 14, 2019
Double Digestion and Dephosphorylation of Plasmid
- iGEM Dusseldorf1
- 1Heinrich-Heine Universität Düsseldorf
Protocol Citation: iGEM Dusseldorf 2019. Double Digestion and Dephosphorylation of Plasmid. protocols.io https://dx.doi.org/10.17504/protocols.io.76shree
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 14, 2019
Last Modified: October 14, 2019
Protocol Integer ID: 28594
Keywords: dephosphorylation of plasmid mix, plasmid mix, incubate at enzyme, plasmid, agarose gel, enzyme, enzymes at suitable conditions load, double digestion, dna
Abstract
Mix the following components gently:
| Plasmid Purified 2016.07.15 | ng/uL | |
| Plasmid DNA Tube X | ||
| Component | Volume (µL) | |
| Sterile MilliQ Water | #VALUE! | |
| 10x FastDigest Buffer | 3.0 | |
| Plasmid DNA | #VALUE! | |
| FastDigest Enzyme 1 | 1.0 | |
| FastDigest Enzyme 2 | 1.0 | |
| FastAP Alkaline Phosphatase | 1.0 | |
| Total Volume | 30.0 | |
| Total ng of DNA | 1000.0 |
Incubate at enzyme suitable conditions
Inactivate the enzymes at suitable conditions
Load on a 1% agarose gel and run for 60 min (110 V).
Cut out digested band (~150 mg) and transfer to pre-weighed tube. Check weight.
Purify the band
Troubleshooting