Question

Q1 A research laboratory identified a gene X of medicinal value in a plant species. You are given a small fragment of DNA containing gene X and the cloning vector pZoom. Maps of the 4 kb cloning vector pZoom and the 10 kb plant DNA fragment are shown in Figure 1 and 2, respectively. The PCR primer pairs (shown in Figure 1) F1/R1, F2/R2 and F3/R3 amplify fragments of 0.1, 0.6 and 0.8 kb, respectively. The antibiotic resistance gene A and gene B code for chloramphenicol and streptomycin resistance, respectively. The three restriction enzymes that cut this vector are BamHI, XbaI and HindIII represented on the map as enzymes I, II and III, respectively. You are given purified DNA of both the vector and the gene X containing DNA fragment at 0.2 µg/ul. Create a recombinant plasmid containing the complete gene X in the provided cloning vector pZoom. a. Design restriction digestion reactions using appropriate enzymes in such a way that you get a final concentration of 50 ng/µl for the digested vector and for the plant DNA (insert DNA) in the reaction. Your digestion should include all components in a 20 µL reaction. All enzymes are supplied with a concentration of 10 units/µL; you may use 1 µL of the enzyme in each reaction. Buffers for each enzyme are available as 10 times concentrated (10X) stocks. b. Generate three ligation reactions with 1:1, 2:1 and 3:1 molar ratios of the insert and the vector DNA. Your ligation should include all components in a 30 µL reaction. Keep the vector amount fixed at 100 ng per ligation reaction. You are provided with 10 x ligase buffer and DNA ligase (0.5 U/µL) to set up your ligations. c. Develop a strategy to select transformants and a quick method to screen recombinants; show your screening method using a figure. Predict the expected results with an explanation. d. Devise a restriction analysis method to confirm the desired recombinant; use a single most appropriate enzyme. Calculate the expected sizes of the restriction fragments from each, the vector and the desired recombinant. e. Devise a PCR strategy to confirm the desired recombinant. Calculate the expected sizes of the PCR fragments from the desired recombinant in a multiplex PCR including all three primer pairs. How will these sizes differ from the same multiplex PCR if the template is vector DNA instead of the recombinant plasmid?

Answer 1

1. the reaction mixture for the restriction enzyme will be as follows using a Vector and gene X DNA 200 ng / micro liter (0.2 µg/ul) stock to final concentration of 50 ng/µl

Genomic DNA - 5 micro liter (1 micro gram DNA for a final concentration of 50 ng/µl in final volume of 20 micro liters )

10X buffer - 2 micro liter

Restriction Enzyme - 1 micro liter (10 U/ul)

Sterile water to make up volume - 12 micro liter

Final Volume - 20 micro liter

2. the insert vector ratio is calculated by the following formula

Nano gram (ng) of vector X size of insert (Kb) / size of vector (kb)   X ratio of (insert / vector ) = ng of insert

suppose the size of vector is 4000 bp (4kb) and three insert fragments of 0.1 kb (100 bp), 0.6 kb (600 bp) and 0.8 kb (800 bp). the the amount of inset for 100 ng vector / micro liter

the three ligation can be set for 100 bp fragment as follows in  1:1, 2:1 and 3:1 molar ratios of the insert and the vector DNA.

1. in 1:1 ratio will be  100 ng x 100 bp / 4000 bp X 1/1 = 10/ 4 ng of insert = 2.5 ng = 1/200 X 2.5 = 0.0125 micro liter

2. . in 2:1 ratio will be  100 ng x 100 bp / 4000 bp X 2/1 = 50/ 3 ng of insert = 05 ng = 1/200 X 5 = 0.025 micro liter

3. . in 3:1 ratio will be  100 ng x 100 bp / 4000 bp X 3/1 = 50/ 3 ng of insert = 7.5 ng = 1/200 X 7.5 = 0.0375 micro liter

the reaction mixture will be as follows for 1: 1, 2:1 and 3:1 molar ratio of inset and vector where concentration vector is 100 ng /micro liter and concentration of insert DNA will be as calculated above, which will be taken from the gene X stock solution (0.2 µg/ul).

	1:1 Molar ratio (I/V)	2:1 Molar ratio (I/V)	3:1 Molar ratio (I/V)
Vector DNA (100 ng vector / micro liter from vector stock of  0.2 µg/ul)	- 0.5 micro liter	- 0.5 micro liter	- 0.5 micro liter
Insert DNA (stock of  0.2 µg/ul)	0.125* micro liter (2.5 ng)	0.025 micro liter (05 ng)	0.0375 micro liter (7.5 ng)
10X ligase buffer	3 micro liter	3 micro liter	3 micro liter
DNA ligase	1 micro liter (0.5 u/uL)	1 micro liter (0.5 u/uL)	1 micro liter (0.5 u/uL)
water to make up volume	25.48 micro liter	25.475 micro liter	25.4625 micro liter
final volume	30 micro liter	30 micro liter	30 micro liter

*Note: the volume of insert is too small to be taken by a micropipette, so the insert stock solution should be diluted before the use.

in the same way, ligation reaction of 600 bp and 800 bp fragment can beset up separately.

Note: the question is solved without the picture of insert and vectors so,which restriction enzyme out of three will be used is unpredictable.

c. after transformation the recombinants can be screened intitialy by antibiotic resistance confered by the gene present in the vector (chloramphenicol and streptomycin resistance) and by blue white selection using IPTG and X-gal on the plate if present in the vector DNA (not picture of vetor is given in the question). the DNA,which contain insert will show white colony and the religated vector containing colonies will be blue on plate.

d. Isolate the plasmid DNA from the blue colonies and digest the plasmid DNA with the same restriction enzyme which was used for the cloning. if correct insert i cloned there it will come out fro the vector that can be seen of the agarose gel and it can be compared with DNA marker. (map of vector and insert not given so unable to predict).

e alternatively the presence of insert can be confirmed by using colony PCR method, in this method the bacterial colony is mixed with ready to use PCR mix with forward and reverse primers flanking the insert. it will amplify the vector along with insert or without insert. when seen on the gel if the insert is cloned the size of the PCR product will be more in comparison to non cloned PCR product.