4. Transformation and plasmid biology (9 points total) You need to transform 40

Question

4. Transformation and plasmid biology (9 points total) You need to transform 40 of XL1 Blue competent cells by adding 10 of a plasmid preparation (concentration: 10pg/uL) that carries a gene encoding -lactamase. a. After heat shock, you add 450 of SOC medium to the transformed cells and place the cells in a shaking incubator at 37°C. Do you add ampicillin (Amp) to this SOC medium? Why or why not? (2 points) b. You centrifuge the soc cell culture, remove 300 of the supernatant and re-suspend the cells in the SOC medium remaining in the tube. You spread 100 of the re-suspended cells on LB + Amp plates and place them in an incubator at 37°C overnight. The next day you count 200 colonies on the plate. What is your transformation efficiency? (5 points) c. In addition to spreading your transformed cells on LB+Amp plates, you also spread an aliquot of your cells on LB only plates. What is the experimental rationale for this control? (3 points)

Explanation / Answer

A. We usually do not add Amp to the SOC medium added immediately afer the heat shock. This is because cells need some to recover from the heat shock and this will provide the bacteria enough time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate.

B.

Transformation efficiency (TE ) = Colonies/µg/Dilution ,

where,
Colonies = the number of colonies counted on the plate
µg = the amount of DNA transformed expressed in µg
Dilution = the total dilution of the DNA before plating

Colonies counted = 200

Amount of DNA transformed = 10* 10 pg {i.e Initially we had taken 10µl of plasmid with a concentration of 10pg/µl }

=100 pg = 100/1000000 =0.0001

Dilution = (10/500) *(100/200)= 0.01

Therefore, TE= 200/0.0001/0.01 = 20,00,00,000 cfu/µg = 2 x 108 cfu/µg

C.

After transformation we need to spread the cells on LB agrar plate without antibiotics as a control. this is done so as to check whether the cells are viable. Luria-Bertani (LB) broth is a rich medium that permits fast growth and good growth yields for many species including E. coli.

Related Questions

Navigate

• Browse (All)
• Browse 4
• Subjects

---

---

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