kwan wrote:First, i would like to say thank u so much for every replies that help me in my first post (in last month ago). all of ur suggestions are helpful for me. now i have some problem again and i need some help from all of u.
i've transformed recombinant plasmid (pHT43 vector 8 kb + DNA 1 kb) into B. subtilis ISW1214 and B.subtilis 1A751. i've got many clones from this transformation but the phenotype of colonies were change and their growth rate are very slow( in both of LB broth and LB plate contained ampicillin) and some clones were not grown after i picked and straeked them on LB+ampicliin plate.
i've ever done a genomic library by using both B. subtilis strains to transform recombinant plasmid (same vector as above) and the the phenotype of clones were change too. it has a same phenotype that i get from PCR cloning i write above.
i don't know why? it may contaminate during the step of preparing competent cell but after i streaked the competent cell,all colony have a same phenotype . it's ok and the phenotype is as same as colony before preparing competent cell. so it was not contaminated,right??? but i don't know why the phenotype of colony were change after transformation.is the size of vector or recombinant plasmid have the effect on phenotype change but i've never seen this problem in transformation of this recombinant plasmid in E.coli.
Could someone here help and explain me about this result ? what i shoud do?i'm so confuse.
Thank you so much for all your help.
amrnegm wrote:i am clooning a protease gene into a bacillus subtilis strain 1012 and i have no expression so far
i have tried to make mini prep from this strain and so far no mini prep
although i can see growth
with a very tubid LB medium
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