Doing work on gene cloning, i want to now that why we insert our target gene into the cloning vector like pMD 19 T Vector, why not /can we just insert our gene into the expression vector directly like PCAMBIA 3301 or any other expression vector to study the gene function?
Hi Fahim
Cloning vectors are used to facilitate gene sequencing, since you can use universal primers, and to ensure you have always a back up stock of your gene (I won't always rely on PCR to obtain your fragment). Some people also like to use a cloning vector because you can use an enzyme from the MCS without having to insert the enzyme site on your primer. However it's possible to clone your gene without cloning first in pMD19T.
You should just be carefu with the enzymes you are using because most enzymes need a small number of nucleotides close to the end so they function (4-6nt). If you count these 6nts plus the 6nts from restriction enzyme that would make a 12nt non homologous sequence which can cause unspecific amplification. But again, pMD19T is not mandatory.
Hi Fahim
Cloning vectors are used to facilitate gene sequencing, since you can use universal primers, and to ensure you have always a back up stock of your gene (I won't always rely on PCR to obtain your fragment). Some people also like to use a cloning vector because you can use an enzyme from the MCS without having to insert the enzyme site on your primer. However it's possible to clone your gene without cloning first in pMD19T.
You should just be carefu with the enzymes you are using because most enzymes need a small number of nucleotides close to the end so they function (4-6nt). If you count these 6nts plus the 6nts from restriction enzyme that would make a 12nt non homologous sequence which can cause unspecific amplification. But again, pMD19T is not mandatory.
Thank you very much Dr. Andre.
can you please tell me what is meant by MCS here .
is there any other way to do sequencing if someone don`t want to use cloning vector.
MCS is multiple cloning site, a sequence of DNA with several restriction enzymes sites.
you can sequence using your primer but in some cases that is not recommended.
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