Lonza provides a plasmid map of pmaxGFP showing the KpnI, NheI, Eco47III, and AgeI restriction sites upstream of max GFP and BglII, XhoI, and SacI downstream. Does anyone know if there are additional sites upstream of GFP? I would like to try and clone my GOI from another expression plasmid into this one due to its small size.
Please refer below Addgene link and you can analyze the sequence for RE
http://www.addgene.org/vector-database/3525/
Thank you for the info, Ganapati. However, I am wondering if the restriction sites are different in their pmaxFP plasmids. According this map I found some of the sites are ordered differently and unfortunately it's the ones I am interested in using. I appreciate the help!
One more entry is in addgene please check and might be useful
https://www.addgene.org/16007/
Dear Sarah,
I have just the same problem than you I want to clone a gene downstream of the GFP sequences in frame to express in mouse cell lines. Have you managed to get the sequences of the MCS? or even better if you have sequenced the vector flaking regions of the GFP? If so would you be able to share with me?
Many thanks
Thanks Sarah for your quick answer, I understand the patent issues, I will check with Lonza before carry on my experiments.
If I get something else about the sequence of the plasmid, I will let you know
Regards
Jose
Hi Sarah, I think the one from addgene (which is pmaxFP-Green-N) mentioned by Ganapati is different with pMAXGFP (yours). And I think those restriction sites written on the map are the only unique restriction sites can be used for single digestion.
Actually I am now facing the same problem with you.
I don't know whether you still work for this now anyway, but I have suggestions for you even if it would take longer time and less chance of success.
You can digest in one of the restriction sites in pMAXGFP plasmid that you want to use and do blunt-ing followed by dephosphorylation. Next, blunt end your GOI (without dephosphorylating it) and insert it into your linear blunted-dephosphorylated pMAXGFP plasmid.
Finally after cloning, you have to screen the correct direction of your GOI by double-digesting the restriction sites inside your GOI and one from pMAFGFP (I will explain further about this if you want) or just read the sequence using your GOI sequence primers. Kind of not so good idea but I would perform what I mention above as well soon. I appreciate if you have better suggestions for me.
I have sequenced Lonza/Amaxa's pmaxGFP plasmid (3486 bp) and am happy to share the sequence with anyone who needs it. Email me at [email protected].
@ Sarah Franco , I urgently need pmaxGFP for transfection experiment. Can you give me some?
Please contact Lonza Scientifc Support, we can provide you details about the pmax-Cloning and pmax-GFP. [email protected]
David Mwin My sincere apologies, I did not receive notification of your post and I have not checked this thread in quite some time (evidenced by my 2-year late response). I do apologize and hope you were able to acquire what you needed. Best,
Sarah
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