This is from a biotechnology class. Please use the photos and read and answer th

Question

This is from a biotechnology class. Please use the photos and read and answer the questions very carefully. Please show all work.

1. Which restriction enzyme(s) should you use to properly cut the DNA fragment? The plasmid?

2. Why does a plasmid contain genes for antibiotic resistance?

3.Draw how the pBR322 plasmid will look once it successfully takes up your gene of interest.

4. You wish to insert this plasmid into a bacteria cell. What method might you use?

5. How will you tell if the bacteria cells has successfully taken up your correct plasmid?

EcoRi Hindili BamHl 5 ...G AATTC...3' 3...CTTAA G...5' Pst EcoRI Tetracycline Resistance Cmail Hind I115 A AGCTT...3' pBR322 (4.36 kb) 3...TTCGA A...5' Bam HI5'...G GATCC...3' Origin 3'...CCTAG G...5' Kmalli 5, C GGCCG 3, 3'...GCCGG C...5 E coll pBR322 plasmid showing restriction sites and resstance genes Pvul Pvull 5'... CAG CTG...3' 3'...GTC GAC...5' PstI 5'...CTGCA G...3' 3'...G ACGTC...5'

Explanation / Answer

1. XMAIII will be used to cut the DNA fragment

2. antibiotic resistance genes act as a selectable marker since when plating is done with antibiotic and agar only transformed cells survive that means having antibiotic resistance genes and other microorganism will die.

3. after treatment with restriction enzyme XMAIII the sequence for the XMAIII is cut and removed which is then taken up by bacterial cell .

4. transformation method is used for inserting plasmid into bacteria.

5. after transformation bacteria are selected on antibiotic plates. bacteria with plasmid are antibiotic resistant so each one will produce colony. which are further used to make large numbers of bacteria and then to produce plasmid or protein

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