Question

Hi! Can someone explain WHY #2 is what it is? I don't understand where those numbers came from. In addition, can you explain where the numbers came from on the bands on the gel in #4? Thanks!

The vector you use contains the lacZ gene and a kanamycin resistance cassette (KanR) BamH1 Lacz Ori EcoR1 ECOR1 Hindlll Vector 20kb Bgll 5kb Pst1 Pst1 7kb Q4. What enzyme will you use if you want to do a blue/white selection screen to determine your construct contains the gene of interest? Why? BamHI because it cuts at LacZ Q5. What is the final size of your completed construct? 20 (vector length) +14.7 (GRE length) +1 (upstream & downstream length) 35.75kb Q6. What color do you expect your successful transformants to be when plated on media supplemented with kanamycin and X-gal? Why? White because they can no longer degrade X-gal

Answer 1

Answer4 . As shown in the vector diagram the BamHI site is in between the lacZ gene that translates for beta galactosidase and in turn help in blue white screening.

Answer 5. The final size include the inserted DNA (14.7Kb) and its constituent like upstream and downstream (1Kb) and vector itself because we are inserting the insert DNA into vector.

So total come 35.75 Kb.

Answer cutting with HindIII there is single band which means that there is only single restriction site in vector.

In case of PstI there are two bands which means that the vector contains two PstI restriction site. As given in diagram size between two PstI site is 7Kb then remain 13Kb will be the size of other bands

Also when we inserted our insert then there are 3 bands sizes are 23kb + 7kb + 5 kb = 35kb which is nearby to our inserted DNA site