You are given two strains of E. coli containing plasmids whose restriction maps

Question

You are given two strains of E. coli containing plasmids whose restriction maps are shown below. You are asked to subclone the gene for alcohol dehydrogenase (Adh) from plasmid B into plasmid A. Unsure of which cloning procedure would be the best, you try two different approaches. You digested both plasmids to completion with Ppul (A/TGCAT) and SnoI (G/TGCAT), isolated the 4 - kb Ppul - SnoI Adh gene fragment and the 3.5 kb Ppul - SnoI double digested vector A. You carried out a ligation reaction containing both isolated DNA fragments, transform E. coli with the ligation mixture, and plated the transformed E. coli cells on medium containing tetracycline. The plates also contain Xgal and IPTG, which allow lthe identification of cells containing the original plasmid A because they turn blue owing to the functional lacZ gene. Colonies with an insertion that interrupts the lacZ gene are white because of insertional inactivation. You followed the procedure in Approach 1 except that you use HindIII (A/AGCTT) instead of SnoI. I. In Approach 1 you obtained many more blue colonies than in Approach 2 Explain why that was so. II. You purified plasmid DNA from white colonies obtained in both of the above approaches Predict the sizes of the resulting fragments when the isolated plasmids are digested with PstI

Explanation / Answer

Ppu I           Sno I

AACGTA ACGTAG

TTGCAT   TGCATC

Ppu I and Sno I will fragment of 4-kb.

Ppu I and Pst I fragment size is 3 Kb.

In approach one if plasmid can insert in right orientation than it will give following fragments;

Digested with Ppu I and Pst I = 3-kb

Digested with Pst I and Sno I= 1-kb

If it incorporate in opposite orientation than

Ppu I and Pst I will give 1-kb fragment.

Pst I and Sno I will give 3-kb fragment.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.