I've seminar on the construction of cDNA library and Genomic library!.So plz clear my doubts n help me!
Thanks for all!
There are some
steps in the construction of cDNA library,They are
* Isolation of mRNA from Tissue.
* Synthesis of first strand of cDNA.
* Synthesis of second strand of cDNA.
* Cloning the double stranded cDNA into a plasmid
* Introduction into the host cells
Here my doubts!
How mRNA is isolated from tissue?
How mRNA is freed from polysaccharide,proteins,etc?
What is polysomal fraction?
How
oligo-dT Chromatography is used to purify the mRNA ?
In
oligo-dT Chromatography,
oligo dT residues which is immobilized on celluose is used, for what purpose it is used?
How a tissue which produces desired mRNA in more amount is selected?
How mRNA is translated invitro? and how polypeptide sequences is obtained?
After translating the desired mRNA in invitro condition,How the polypeptide sequences are checked in the isolated mRNA ?How they know the protein sequence of that mRNA sequence in prior ? how it is made?
How mRNA fraction is copied into the 1st strand of DNa as single strand?
function of Reverse transcriptase?
what is
oligo-dT?
how reverse transcriptase helps in adding appropriate nucleotides to oligo-dT primer ?
how mRNA is denatured in the process after the synthesis of first strand of cDNA synthesis?
how nuclease s1 cleaves the hair pin loop?
what is klenow fragment of DNA polymerase one ?
why especially E.coli k-12 strains are used ?
Structure of Pbr 322,What is psI site ?
Why rDNA should be cloned tin the PSI site of pBR322 ?
how ampR gene of pBR322 is deleted while Gene of interest is cloned
Plz help as soon u can?
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