Q: Which antibiotic would you use to select for Agrobacterium tumefaciens containing a kanamycin resistance gene, and what is the working concentration?

Kanamycin at $$50 \mu\text{g}/\text{mL}.$$

Question 1

Which of the following is a critical step to ensure the functionality of antibiotics and acetosyringone in the ATTA protocol?

Accepted Answer

Add antibiotics and acetosyringone after autoclaving the media, using sterile technique.

Question 2

During the Agrobacterium transient transformation assay (ATTA), why is the OD600 measurement important, and what is the ideal range for bacterial viability?

Accepted Answer

OD600 measures bacterial density; ideal range is 0.6-2.0, preferably 0.8.

Question 3

A student is preparing infiltration medium for ATTA. If they need 75 mL, how much sucrose should they add, given the recipe calls for 20 g/L?

Accepted Answer

Add $$1.5 	ext{ g}$$ sucrose.

Question 4

In the rapid boiling miniprep protocol, what is the purpose of adding RNAse A to the STATT++ buffer?

Accepted Answer

To degrade RNA and ensure only DNA is isolated.

Question 5

Which antibiotic would you use to select for Agrobacterium tumefaciens containing a kanamycin resistance gene, and what is the working concentration?

Accepted Answer

Kanamycin at $$50 \mu	ext{g}/	ext{mL}.$$

Question 6

Why is it important to use a negative control in a colony PCR experiment?

Accepted Answer

To confirm that any observed amplification is not due to contamination.

Question 7

A student is performing electroporation of Agrobacterium. What is the correct sequence of steps after adding plasmid DNA to competent cells?

Accepted Answer

Incubate on ice, electroporate, add SOC medium, incubate at 28°C.

Question 8

In SDS-PAGE followed by Western blotting, what is the primary purpose of the blocking step with TBST and milk powder?

Accepted Answer

To prevent non-specific binding of antibodies to the membrane.

Question 9

If a student wants to check for off-target effects of a CRISPR sgRNA in Nicotiana benthamiana, which tool and reference genome should they use?

Accepted Answer

Cas Off Finder with N. benthamiana genome v1.0.1.

Question 10

During DNA isolation from plant tissue, why is isopropanol added after the extraction buffer and NH4Ac?

Accepted Answer

To precipitate DNA from the solution.

Question 11

A student is running a PCR with a total volume of $$25 \mu	ext{L}. If $$they use $$1 \mu	ext{L} of $$each primer (10x diluted to $$1 \mu	ext{M}$$ final), what is the final concentration of each primer in the reaction?

Accepted Answer

$$1 \mu	ext{M}$$

Question 12

In the post-transformation '3-traps rocket' protocol, why is it important to create a collection plate after picking colonies?

Accepted Answer

To ensure you can re-inoculate the same colony later for further experiments.

Question 13

What is the main function of SDS in SDS-PAGE?

Accepted Answer

To denature proteins and impart a uniform negative charge.

Question 14

A student is preparing a restriction analysis. Why is it important to check the optimal conditions for the chosen enzyme?

Accepted Answer

Because some enzymes require specific buffers or additives like BSA for activity.

Question 15

In the ATTA protocol, what is the purpose of acetosyringone in the infiltration medium?

Accepted Answer

To induce virulence gene expression in Agrobacterium, enhancing T-DNA transfer.

Question 16

Why is it necessary to use different gloves when infiltrating leaves with different Agrobacterium genotypes?

Accepted Answer

To prevent cross-contamination between genotypes.

Question 17

A student observes fluorescence in the infiltrated area of a leaf after ATTA. What does this indicate?

Accepted Answer

Successful expression of the transformed gene, such as GFP.

Question 18

In the gel/PCR cleanup protocol, why is it important to use a kit like the Wizard SV Gel and PCR Clean-Up System?

Accepted Answer

To efficiently purify DNA fragments from gel or PCR reactions for downstream applications.

Question 19

When preparing competent Agrobacterium cells for electroporation, why must the cells be kept on ice until transformation?

Accepted Answer

To maintain cell viability and prevent DNA degradation.

Protocols and Techniques in Plant Gene Editing and Molecular Biology

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