Upcoming lab practical and I need help with the following, Biotechniques- Clonin

Question

Upcoming lab practical and I need help with the following,

Biotechniques- Cloning

1. Be able to read a restriction map

2. Give a plasmid and insert be able to make different combinations of plasmids

a. know the enzymes used for cloning and what they will do.

i. Restriction enzymes, ligase

b.Know the requirements of a plasmid and what they do

i. Antibiotic resistance

ii. Ori

c. know what the other elements are the were in pGLO

d.Understand what a tagged protein is and how that effects properties of fusion protein

i. N-term vs C-term

ii.size

iii.requirements to visualize -UV

Thank you. Will give a thumbs up. (I would like to read a simple, straight to the point explanation. Make sure your answer is well organized) THANK YOU.

Explanation / Answer

1. Restriction map is a map of known restriction sites within a sequence of DNA. It requires restriction enzymes to break DNA into pieces.

2. a. The restriction endonuclease are the sites within genes for selectable markers.

b. pBR322 is the most popular and moste widely used plasmid vector with size of 4362 base pairs.

c. It has to selecteable markers, 1. Ampicillin and tetracycline restance, lacZ gene are markers with 12 restriction endonucleases.

d. It contain the origin of replication of pMB1 and the rop genes.which allows initiation of replication to encode plasmid copy number.

e. The AmpR gene contain two promoters P1 and P3 for beta-lactamase. P3 is a natural promoter and Pq created by the ligation of two different DNA fragments.

f. pGLO is bacterial transormation plasmid for regulating expression of GFP (Green fluorescent protein)

g. The enzyme ligase joints two DNA strands together by catalyzing the formation of a phosphodiester bond.

3. a. Tagged progeins are peptides sequences that are attached to protein to facillitate easy detection and purification of expressed proteins.

b. C-term and N-terms are location of tags. Either the N-term or C-term side is removed to verify correct protein processing.

c. Protein crystallization experiments with UV light enables crystallographers to differentiate between crystals of protein and salts.

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