pART27 vector

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Vinny
Vinny's picture
pART27 vector

I have a problem with cloning pHannibal cassette in pART27vector....I cut phannibal cassette and open pART27 with NotI and seems to be OK....after i treat the pART27 with CIAP, extract all the two bands from agarose gel...ligate and cloning with electroporation, the ligation in E.coli DH5 alfa...after i select on antibiotic Kanamicin 50ug/ml...but not works....There's anyone that can help me????

Tony Rook
Tony Rook's picture
Vinny:

Vinny:

See my reply to your similar post in the Plasmid/Cosmid/BAC/PAC topic of the molecular biology forum:

http://www.scientistsolutions.com/t3569-Problem+with+pART27+vector.html

Have you tried lower concentrations of antibiotics during your selection step. Also, have you confirmed any surviving culture after your electroporation step. It may be possible that your culture is no longer viable. In such case, you may need to repeat this cloning step.

Vinny
Vinny's picture
Tony Rook

Tony Rook

Thank you very much for the reply.....
I try to apply your suggestion.... let we see...

Vinny
Vinny's picture
I try with go down Kanamicyn

I try with go down Kanamicyn antibiotic concentration 25ug/ml but nothing....No colony....I have also a negative control with ciapped vector in ligation without fragment and i not have colonies.....the same bacterial concentration on LB without antibiotic show a good growth and the trasformation with the uncutted vector work.....
It's very strange....my new idea is to not purify the vector on agarose gel after NOT I and Ciap treatment but purify or with Kit for purification of PCR products or with phenol-chloroform because probably there are some problems with residual presence of agarose or ethidium bromide ....i don't know

kamalinder
kamalinder's picture
Vinny wrote:I have a problem

Vinny wrote:

I have a problem with cloning pHannibal cassette in pART27vector....I cut phannibal cassette and open pART27 with NotI and seems to be OK....after i treat the pART27 with CIAP, extract all the two bands from agarose gel...ligate and cloning with eclectroporation, the ligation in E.coli DH5alfa...after i select on antibiotic Kanamicin 50ug/ml...but not works....There's anyone that can help me????

friend i think you should select the pART27 binary vector on streptomycin (25 ug/ml)/or spectinomycin (50-100 ug/ml) as in the original paper of pART27 these two antibiotics are shown in the map for bacterial selection.

ayudhee
ayudhee's picture
I have lost sequence of

I have lost sequence of pART27 coz my laptop was stolen. There's anyone can email me the sequence of pART27?? (my email eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%64%69%6e%69%5f%61%79%75%30%33%40%79%61%68%6f%6f%2e%63%6f%6d%22%3e%64%69%6e%69%5f%61%79%75%30%33%40%79%61%68%6f%6f%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))).

Abeer
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grow pART27 on Spec100ug/ml.

grow pART27 on Spec100ug/ml. pART27 does not grow on Kanamicin

aquarium
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Dear Vinny,

Dear Vinny,
 
Myself from National Chemical Laboratory Pune, India. I came across your mail id regarding some of your query about pART27 vector.
I need to carry out some gene down regulation studies in plant system. In this regards can you let me know from where did you get the pART27 binary vector. If possible can you provide me the same.
I will be very thankful and oblidged if you can help me in this regards,
 

 
            

emily
emily's picture
Im from Multimedia University

Im from Multimedia University, Malaysia. I need to carry out some gene upregulation study in plant system. Could you let me know where i can get the pART7 and pART27 vector?Could you provide me? TQ

Yulia
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I also saw the map for pART27

I also saw the map for pART27 in Andrew Gleave's paper. But I don't have the sequence. Do you get sequence?

manueliux_06
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Hi

Hi
I´m working with pHANNIBAL and i have my cassette of expression but I have not the pART27 vector. Can you help me to get it the bynary vector pART27? Could you send it to me? or Where are can buy?

hana
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 maybe you need to change

 maybe you need to change your antibiotic. use ampicilin instead of kanamicin