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Confirming insert????

insert

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#1 Gunalan

Gunalan

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Posted 01 December 2011 - 06:13 AM

Hii All
I have done transformed the ligated product into the E coli DH5 cells the colonies were growing on the LB agar with ampicillin plated but when i inoculate them onto LB broth with ampicillin for plasmid isolation they were not growing at all.in most of the tubes I use 15 ml test tubes with 5 ml broth and grow them in shaker.
what could be the problem???


Later on i got Some growth in some broth from them i isolated plasmid and Subjected it to RE digestion iam getting almost double sized insert I cold not understand what is happening

I use BamHI as one of the enzyme and digest for 2 hr.
can anyone suggest me on this regard so that i can confirm my insert .


Thanks
Gunalan

#2 GNANA

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Posted 01 December 2011 - 11:48 AM

did you put proper controls to make sure your plates and competent cells were free of contamination?
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....





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