Exploring Recombinant DNA Technology

Describe a scenario where a gene cloning technique fails. What steps would you take to troubleshoot the issue?

• A gene cloning technique fails due to unsuccessful transformation of host cells.

• The gene cloning technique fails due to the presence of antibiotics in the growth medium.

• The gene cloning technique fails because the plasmid is too large to be taken up by the host cells.

• The gene cloning technique fails due to excessive temperature during incubation.

You are optimizing a PCR reaction but consistently get non-specific bands. What adjustments could you make to improve specificity?

• Use a longer extension time

• Decrease primer concentration

• Increase annealing temperature and optimize primer design.

• Add more template DNA

Explain the factors you would consider when selecting a vector for cloning a large gene. What challenges might arise?

• Consider vector size, origin of replication, selection markers, promoter compatibility, and cloning site; challenges include transformation difficulty and insert instability.

• Vector size is irrelevant for large genes

• Only consider the cost of the vector

• Promoter compatibility is not a factor in cloning

You notice low transformation efficiency in your bacterial cells. What potential causes could you investigate to improve this?

• Switching to a different bacterial strain without testing

• Investigate cell competence, DNA quality, transformation protocol, growth conditions, and recovery media.

• Increasing incubation time only

• Using outdated plasmids

During gel electrophoresis, your DNA bands appear smeared. What could be the reasons for this, and how would you address it?

• Incorrect sample type used

• Inadequate staining time

• Smeared DNA bands can be caused by overloading, DNA degradation, improper gel preparation, high voltage, or unsuitable buffer.

• Using too low a voltage

You are sequencing a DNA fragment, but the results show poor quality reads. What troubleshooting steps would you take?

• Use outdated sequencing technology without adjustments

• Check sample quality, verify library preparation, adjust sequencing parameters, consider different platforms, analyze data for errors.

• Ignore the results and proceed with analysis

• Increase the sample size without checking quality

Imagine you are cloning a gene but the insert does not ligate into the vector. What could be the possible reasons and solutions?

• The vector is not compatible with any gene.

• The ligase works better at lower temperatures.

• The gene is too long to clone.

• Possible reasons include incompatible ends, low concentrations, inactive ligase, incorrect buffer, or inhibitors; solutions involve ensuring compatibility, adjusting concentrations, checking ligase activity, using the right buffer, and purifying DNA.

You have optimized your PCR conditions, but the yield is still low. What additional factors could you explore to enhance yield?

• Explore primer design, template quality, enzyme concentration, cycle number, reaction volume, and additives.

• Reduce the number of primers used

• Increase the temperature of the PCR reaction

• Use a lower quality template

Discuss the importance of antibiotic resistance markers in vector selection. What issues could arise if they are not properly utilized?

• Antibiotic resistance markers are essential for ensuring the effective selection of vectors in genetic engineering and microbiology.

• Antibiotic resistance markers are only relevant in human medicine.

• Proper utilization of antibiotic resistance markers is irrelevant to microbiology.

• Vector selection does not impact the effectiveness of genetic engineering.

You are trying to transform E. coli with a plasmid, but the cells are not taking up the DNA. What could be the underlying issues?

• Possible issues include inadequate heat shock, poor plasmid quality, cells not in optimal growth phase, or inefficient transformation protocol.

• The plasmid is too large to enter the cells.

• E. coli cells are naturally resistant to all plasmids.

• The transformation was performed at room temperature.