Hello
for cloning my gene of interest into a plasmid, i placed BglII and ApoI sites into the ends of my gene but there is a EcoRI site in my gene sequence. i want to know whether ApoI will cut restriction site of EcoRI?
thanks
I use a software program called Serial Cloner to do quick checks on restriction sites, translation frames, etc. That being said, I believe EcoRI cuts at GAATTC versus ApoI which cuts at 5'-R AATT R'-3' so if your R and R' are G and C, respectively, then it they will both cut that site.
BgIII has an optimal digestion temp of 37C, while ApoI is 50C, it's not optimal but it should cut fine, NEB recommends using 3.1 buffer. They have a very useful tool over at https://nebcloner.neb.com to help you sort out double-digestions, buffers, protocols, etc. for restriction cutting.
Just to amend Vincent's post above for clarity :
ApoI cuts at 5'-(R)AATT(Y)-3' using conventional base codes for Purine (R) and pyridamine (Y) respectively- so EcoRI sites are indeed targeted by ApoI - but GAATTT and AAATTC would also be targeted.
More pertinently, Maliheh Davari - why did you use ApoI as a restriction site? - was there a specific reason for choosing this enzyme rather than another more conventional enzyme?
Thanks for all answers
about Robin's question must be mentioned that actually i faces with a complex cloning in that i have a vector which can only cut with BamHI and ApoI versus a DNA fragment flanking with BamHI and XhoI sites. unfortunately this DNA fragment contain some enzyme sites such as BamHI, EcoRI and XbaI. For cloning this DNA fragment into my vector, i have to design primers with BglII and ApoI overhangs (BglII creates sticky ends compatible with ones related to BamHI). that is why i have to use ApoI site. although if ApoI and EcoRI are able to recognize and cut the sites of each other, i will use EcoRI overhang instead of ApoI in primer designing.
Glad to hear your ideas
sincerely
Maliheh
ApoI will cut EcoRI sites, but EcoRI will not cut all ApoI sites. If there is an issue with internal cleavage, one option would be to perform a partial digest - cut initially with BamHI in a larger volume (i.e. 50 / 100 micrometers), then add a smaller than normal amount of EcoRI and start the timer. Remove aliquots to a stop buffer (EDTA / SDS / Tris / on ice) to prevent further digestion every few minutes, then run the digestion time points out on agarose - over time you should see full length band becoming two bands as the internal EcoRI cuts. I then take the full length band from several lanes and hope that this has enough sticky ends for cloning use.
Good luck!
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