plasmid cloning in E.coli - (May/07/2007 )
Hi everybody!
I'm quite worried, because I have loads of questions but I can't ask anyone here in lab, so I hope to find on the forum someone so kind to share his experience.
I've bought the plasmid and it arrived blotted on paper. The producer suggests to elute it and then to clone it in E. coli to multiply it.
I will use Promega JM109 E. coli cells, so my very first question is: how can I understand if it would be possible to perform the blue/white screening? What else screening method can I refer to?
I've checked my plasmid and it seems to have a lacZ alfa segment, with a polylinker sequence inside.
Will it be sufficient to perform alfa-complementation? Can I be sure that the bacterial strain and the plasmid are compatible?
Any advice, theorical explanation, suggestion...anything would be of great help!
Thank you!
Ila
ila i dont have any experience to elute DNA from blotted paper but so far i know its very easy and simple.
may be it is just add the blotted paper in TE buffer and collect suparnatant and take some supernatant for trasnformation in e. coli and check by miniprep as usual.
may be you will get more advice, explanation and suggestions from experience people, but here i just express my opinion and i also want to check my thinking with others
I will use Promega JM109 E. coli cells, so my very first question is: how can I understand if it would be possible to perform the blue/white screening? What else screening method can I refer to?
No worries about transforming the plasmid in JM109. Blue/white selection? Have you clone anything yet to your plasmid? I mean is the lacZ gene is being disrupted? If it is just the parental vector, my answer for blue/white selection is no. Well, antibiotic selection is pretty common. As long as the colony has the vector with the antibiotic resistance gene, I am quite sure the colony has the vector. If you have the specific primer for the plasmid, it will be better still.
Hope this helps.
