I've tried five times now, using PCR to amplify and add 4 different combinations of restriction sites to try to insert into a pCDNA3 derivative. I've had great success with the vector with other inserts during this time but get no GFP inserted. Any suggestions? All I can think of is some structure in the GFP DNA that inhibits ligations. Perhaps switching to a different fluorescent protein is necessary.
Sometimes, restriction digest of the termini of a PCR product can be inefficient. I would agree with Swami above (for a different reason), and clone into a cloning vector like pGemTEasy first, and the digest and clone again. We do most of our cloning this way - takes an extra for days, but works much better.
Cheers,
Steve
I would be surprised if it has anything to do with DNA structure. Some restriction enzymes do not work, if the restriction site is at the extreme end of PCR product. Therefore, while designing primers make sure that restriction sites are 5-6 nucleotides away from the termini. I would agree to the idea of cloning your insert in pGEMTeasy vector.
Can you clarify exactly what the problem is? Are you getting a PCR product and are just unable to restrict and insert it? Or is the PCR also not working?
Thanks for responses to date. To clarify, I have many years experience with sub-cloning successes and this is unusual. The PCR reactions work well, the primers include a 10 bp GC rich clamp at the ends, to promote complete cleavage of RE sites. The vector is good as other inserts, prepared in the same way (PCR), ligate in efficiently. The only thing I can conclude is that at least one end of the eGFP PCR product has a structure that inhibits RE digestion and thus cannot ligate. I have encountered other cases where sequencing confirms the presence of an RE site that does not cleave, both in plasmids and in PCR products. Curious if this is a known issue with eGFP.
Apologies Richard, clearly you do have a lot of experience - should have looked at your profile first.
I am sure I, and probably others in our lab, have cloned eGFP, and I can't recall there being any issues that I can remember... it was a long time ago for me at least.
We do tend to sub-clone by A-tailing first before going into an expression plasmid (as my first post suggested). Although I have directly cloned from digesting the PCR product, we have changed our lab protocol to encourage our students to do it this way as it saves time in the long run. One benefit you might find by doing it this way is that you will definitely know whether your restriction digest has worked, as it will be clearly differentiated from the backbone after digestion, rather that hoping the tiny termini have been trimmed off.
Hope it is a simple fix,
Steve
I had some problems with subcloning GFP into TOPO TA vectors last year. A colleague advised me to add glucose to plates (I used 0.5% final conc.) and subsequently glucose in the overnight cultures (0.2% final conc.) prior to miniprepping. It worked a treat for me.
Good luck
Maia