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difficult to find the transformates containing insert DNA - ligation, transformation (Sep/14/2004 )

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Hi, guys,
I meet a problem with inserting a gene, GFPuv(green fluorescent protein) to a plasmid pUT-Tn5. First I cut this gene together with its promoter, Plac from the plasmid pGFPuv and ligated to auxiliary vector, pUC18NotI by blunt-end liagtion. It gives me very good results with quite high expression in E.coli JM109. i can see it clearly under uv light. This really gives me much confidence in molecular biology since i am a beginnner here. However when I cut this gene from the auxiliary plasmid by RE NotI and ligate it to pUT-Tn5 which also containing a NotI site, all the colonies I get by using the ligation products for transforming doesn't show much fluorescence under uv light as i expect. Didn't I ligate the insert DNA to the plasmid? I use the same protocol as first time I use for ligation to the auxiliary plasmid. I even tried using CIP which I didn't use for auxilary plasmid, it doen't work either. Everybody saids that cohensive ends works much higher efficienct than blunt ends, but I just get the opposite results. Blunt end ligation works for me, sticky end doesn't work. I don't know why. Is it the expression of GFP protein too low that I can't screen the transformates by uv light?But why it works first time with pUC? and I think it should also work this time since the strains is not big different, JM109 and DH5alpha √ępir? Can anybody give me any suggestion? I will be very grateful for that. Thanks very much! smile.gif

-hubeimm-

Maybe your insert is ligated in an inverted position, I mean the 3' end at the promoter site?

-biotech-

Thank you very much for your reply. But I think the chances of get two direction of ligation of the insert should the same. So if there is invert ones there should the right ones. rolleyes.gif

-hubeimm-

hi, guys,
I get some new infomation about my experiment now. but i still need you guys help. I did the negative control. I used the dephosphorylated plasmid and cutted plasmid to transform the E. coli and I get zero colony. Then I used this dephosphrylated plamid to ligate my foreign DNA (GFP gene) and then did the transfomation again. I also get zero colony. I couldn't figure out what the problem is. Is it the T4 ligase doesn't work? I check the ligation mixture on the gel and it gives me different picture from without ligase. Does the transfomation efficiecy too low? But i did get colonies just by emply plasmid. I use TSS transfomation. Or if my gene too toxical to my bacteria? We have some guy in our lab who get it tranformed to the same E. coli. What would you say?

-hubeimm-

gfp gene is amplified by PCR?
your oligos are phosphated?
Because if not, and you dephosphated your vector....they are no chance to meet...smile.gif (and the phosphodiester bond works out.)



Bye,
B. smile.gif smile.gif

-blasko-

thank you blasko
I am sorry i don' t understand your meaning. I cut the gfp band directly from the gel and i didn't phosphoylate this fragment. I only phosphorylate my vector. Is that wrong?

-hubeimm-

Did you amlify GFP gene by PCR or
did you cleavage GFP gene from a plasmid by restr. enzyme?


B.

-blasko-

In addition to, if you made PCR, did you order phosphated oligos?

-blasko-

No, Blasko,
I use restriction enzyme to digest my plasmid and then run the gel and isolate from the gel. Do I need phosphorylate the fragment?
thanks

-hubeimm-

if your insert is from a restriction digest, it is already phosphorylated, so you don't have to extra phosphorylate the insert DNA . should be ok.

mike

-jadefalcon-

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