Hi,
I am working with Invitrogen Seamless cloning which requires 200 ng/ul insert DNA fragment. Protocol recommends to use restriction enzyme to digest pre-cloned plasmid and then elute specific fragment (3 kb in my case). However, most of time, commercial gel elution kit can give me elutent with a low concentration and yield. Does any one know to how make it work? I tried to release DNA fragment in TAE solution as the gel was loaded in a plastic bag under gel electrophoresis (then use PCR purification kit to elute DNA). Even though it worked better than conventional gel elution, the concentraction and yield are still low, and it also requires a lot of plasmid. Additionally, non-specific bands were included in elution product. Has anyone carried out a similar experiment and is willing to share a protocol? I also guess magnetic beads can work, but I am still looking for a protocol.
Thank you in advance.
Andrea
Today, gel isolation of a fragment to be cloned is somewhat clumsy. Even if you get enough DNA for cloning, impurities from agarose significantly decrease efficacy of cloning. If possible, avoid gel purification.
Combination of PCR amplification of insert (or even vector) and Gibson assembly (or similar) is far more reliable and fast. Martin
How long are you digesting? I found that it was better to do the digestion for 2 hours. I also completed the digestion reaction in the cloning vector first then digested the eluate with the second RE in a 10ul reaction (instead of the recommended up to 50ul). Run the digested DNA on 0.8% gel using low melting point agarose. I purified the DNA via DNA gel extraction using an Enzo Agarose Gel Extraction Kit (Spin-column based DNA cleanup from agarose gels) and eluted in 30ul of ddH2O (45.2ng/ul is what I ended with). I encountered problems when sequencing later if I used any sort of elution buffer. On the other hand, I do agree Gibson Assembly is reliable, which requires 100ng of Destination and 100ng of Entry for the ligation reaction. Restriction Enzyme cloning using T4 ligase only requires 20ng of digested DNA. I am unfamiliar with the Seamless Cloning, but if you could share your protocol I can try to help.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology