compare pEGM T easy vector and TOPO TA cloning - (Nov/07/2007 )
anyone knows the advantage of TOPO TA cloning system to pEGM T easy vector?
I thought you did not need IPTG and X-gal. Any advantage on forming more positive colonies? or something else?
I thought you did not need IPTG and X-gal. Any advantage on forming more positive colonies? or something else?
It depends on which system you choose. There is a TOPO TA vector that contains a lethal gene. An insert will disrupt the gene and colonies will be able to grow, if a plasmid does not get an insert it will die. So any colonies that grow will contain an insert. I'm not sure of the name or the exact details but I remember coming across it when I was choosing a TOPO TA vector - Check the invitrogen website.
The TOPO TA vectors have an topisomerase attached so all you do is incubate your insert and the vector with a salt solution. Using the topoisomerase enzyme as opposed to DNA ligase is meant to be quicker and more efficient. I use TOPO TA pCR2.1 vector, which uses the blue white screening system - so you do need x-gal. It works really well. I find that it is more efficient than pGEM-T easy and that I get more white colonies with my insert. In addition, the cloning process can be quicker than the pGEM system. You can choose to use Mach1-TiR cells which grow quicker and the ligation only takes 30 minutes.
I still use pGEM-T easy for some basic routine cloning since it is cheaper than TOPO TA pCR2.1.
- You don't need to use IPTG with TOPO TA.
Hope this helps!!
I thought you did not need IPTG and X-gal. Any advantage on forming more positive colonies? or something else?
It depends on which system you choose. There is a TOPO TA vector that contains a lethal gene. An insert will disrupt the gene and colonies will be able to grow, if a plasmid does not get an insert it will die. So any colonies that grow will contain an insert. I'm not sure of the name or the exact details but I remember coming across it when I was choosing a TOPO TA vector - Check the invitrogen website.
The TOPO TA vectors have an topisomerase attached so all you do is incubate your insert and the vector with a salt solution. Using the topoisomerase enzyme as opposed to DNA ligase is meant to be quicker and more efficient. I use TOPO TA pCR2.1 vector, which uses the blue white screening system - so you do need x-gal. It works really well. I find that it is more efficient than pGEM-T easy and that I get more white colonies with my insert. In addition, the cloning process can be quicker than the pGEM system. You can choose to use Mach1-TiR cells which grow quicker and the ligation only takes 30 minutes.
I still use pGEM-T easy for some basic routine cloning since it is cheaper than TOPO TA pCR2.1.
- You don't need to use IPTG with TOPO TA.
Hope this helps!!
This is Very helpful. Thanks for sharing your experience.
My friend suggested me to use PCR4 TOPO vector as he got more success using this guy. Do you know the difference between pCR2.1 and PCR4 TOPO vector?
Hi!
The pCR4 vector is for blunt ended cloning and the pCR2.1 is for TA cloning (i.e. PCR products with an A overhang).
The pCR4 vector is for blunt ended cloning and the pCR2.1 is for TA cloning (i.e. PCR products with an A overhang).
I think PCR 4 is also for TA cloning. They have PCR-blunt for blunt cloning.
Vector maps of both vectors:
http://www.invitrogen.com/content/sfs/vect...cr4topo_map.pdf
http://www.invitrogen.com/content/sfs/vect...2_1topo_map.pdf
Check the manuals from the kits they come with.
I only have experience with TOPO cloning and not the other one, and I like it. I use TOPO XL (for larger fragments but I have used it for smaller also because of its existense in the freezer).
A little off topic, but why the white blue screening? Is that better you think?
I first LB-agar/antibiotits plate to grow it and then I use pcr (a dip of bacteria directly into the tube) to confirm the presense of my insert.
If you plan to sequence the insert of pCR2.1, please be aware of this:
Resolving a DNA sequencing artifact associated with topoisomerase I generated clones in the plasmid pCR®2.1
Andrew A. Forbes, Thomas H.Q. Powell, Neil F. Lobo, and Jeffrey L. Feder
BioTechniques Vol. 42, No. 4: pp 458-462 (Apr 2007)