Question

1. ) Describe the basic process of producing a plasmid containing the gene for factor X, a gene from human cells that codes for clotting factors. Include all the sequences that might be needed in the promoter as well as the enzymes and steps involved in making the plasmid.

Answer 1

Making plasmid.

1) Isolate human Dna

2) isolate the gene of interest by sequencing it and finding the restriction enzyme sites near it.

3) Use restriction enzyme for cutting the dna and isolating the required gene of interest.

4) run on agarose gel to extract the dna out.

5) Use PCR for amplifying the gene of interest usi g proper primers - the enzymes include - Taq polymerase , Primase, primers , dNTPs in equal ratios. Buffer, Ions like Na+, Mg2+, Ca2+ etc.

6) isolate the amplified dna and cut the dna again with a particular restriction enzyme. For getting specific region on dna of the gene.

7) cut the plasmid vestor with the same restriction enzyme to get the same sticky ends.

8) incubate the vectors and gene of interest togethor along with dna ligase enzyme.

9( The vector will be prepared carring the gene of interest.