Entry Level: BioTech

Preparing DNA for PCR

Simulation vetted by professionals from CMDOs in Texas

Industry Job Title:

Research Lab Technician

Associated Simulation Library:

Background

As a research lab technician, you are responsible for carrying out specific tasks or experiments. One task, isolating DNA from cells, is quite common across labs that specialize in molecular biology and genetics and is even necessary for computational biology. These experiments span many organisms—you might work with DNA from humans, mice or zebra fish or even plants and bacteria. Being proficient in DNA isolation and handling is a well-sought after skill. Depending on the lab, your job may perform these isolations independently or you might work on a team.

The source of DNA can vary based on the experiment and the lab, but the concepts and techniques for isolation are generally the same. This task is extremely important though, and is often a crucial step in understanding genetics-based questions. For example, successful DNA isolation is necessary for:

  • Confirming the presence and location of specific genes within a given genome 

  • Confirming the successful deletion of addition of genes into a genome

In humans, DNA isolation can be performed to check for the presence of cancer-related genes to help design cancer treatments and therapies, making it important for cancer patients and researchers. In clinicals, DNA isolations may be performed on blood samples collected from patients. In plants, DNA isolation can be performed to find where drought-related genes are located to help build more durable crops, impacting everyone on the planet. In bacteria, DNA isolation can be performed to design plasmids (or small circular DNA that carry genes of your choice) used to introduce new genes in other organisms like plants, for example. Thus, bacteria DNA isolation is a powerful tool for genetics across fields. 

Considering this, the ability to isolate DNA is extremely important and has the potential to impact research of all kinds. Ready to practice a DNA isolation task?

  • deoxyribonucleic acid; molecule that carries the genetic information of nearly all organisms

  • polymerase chain reaction; amplification technique used to make several copies of nucleic acids

  • kits that include reagents and instructions for isolating DNA

    Nanodrop - spectrophotometer used to take highly accurate analyses of DNA, RNA, and protein (Boston Uni).  

  • DNA quality is read using a spectrophotometer. A common type of spectrophotometer is a Nanodrop because it requires very small volumes of your sample. 

    Nucleic acids have absorbance maxima at 260 nm. The ratio of absorbance at 260 nm and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm (Lucena-Aguilar et al.). 

    In other words, when you read the DNA, there will be a 260/280 ratio, the closer the reading is to 1.8, the cleaner the sample.

  • For most reactions, you only need very small amounts of DNA. Sometimes precise amounts of DNA are not needed. Simple dilutions, like 1:10 or 1:100, can be used to achieve low enough concentrations. Some experiments require precise amounts of DNA. To do this, you need to do math to calculate the proper amount. 

    For example: 

    If you have isolated 200ng/ul of DNA and only need 5 ng for an experiment: 

    Calculate 50/200 to know how many microliters (ul) of your isolated DNA to add. 

    50/200 = 2.5 (add 2.5ul of the isolated DNA to your reaction)

Common Vocabulary

(expand each entry for full definition)

The Process

  1. Get rack of test samples of whole blood (in the purple top tube)

  2. Transfer samples to the lysis tubes

  3. Isolate DNA Lyse cells (get DNA from WBC), Qiagen kit blood and tissue (send pictures of the columns, column is cloudy or red; visually inspecting tubes to look for things that look different; take from trouble shooting steps in the protocol)

  4. Set the equipment to the right settings to centrifuge the sample

  5. Complete Purification steps DNA, RNAse treat

  6. Quantify the DNA (look at yield and concentration) 

  7. Record and Review

  8. Aliquot the DNA

  9. Store in 4C and -20C

Resources

Foundational knowledge on DNA & DNA isolation:

Foundational knowledge on PCR and gel electrophoresis:

The Exercise

You are an intern who has received purified DNA samples from blood cells for testing. You need to set up a PCR to check for the newly discovered howdy gene. Before doing the PCR, you need to select the DNA samples with the best quality and calculate how much of each to add to the reaction. 

Task 1 - Select the Samples with the Best Quality

This spreadsheet provides information on quality readings for your 3 samples: the control sample, sample A, and sample B. You isolated 3 replicates for each. Select the rep with the best quality for each sample. 

To determine quality of the sample - look at the ratio of absorbance at 260 and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm.

Task 2 - Determine the Volume of Each Sample to Add to the PCR

For each final sample you chose, calculate how much of each is needed for a final concentration of 200ng/ul. You should have 3 separate calculations for 3 separate reactions. 

Task 3 - Calculate Final Volumes for the PCR Reaction

Eacn reaction will use 5ul PCR buffer, 1ul DNA polymerase, the rest is made up of isolated DNA and water. The final volume should be 10ul. You’ve already calculated how much of each is needed for 200ng DNA. Now, back calculate to see how much water to add to each reaction.

Control sample:

Sample A:

Sample B:

Once you’ve calculated how much is needed for each reaction, you’re ready to run your PCR! Great job! 

Helpful Videos

Watch the process of PCR

Watch the process of running the PCR-amplified DNA on a gel

Deliverables

After isolating the DNA, you may show the research advisor your notebook or a copy of the DNA quality spreadsheet. If you’ve selected the samples you will move forward with and have done the calculations, you may show those as well.

Sometimes you are expected to show the gel electrophoresis which displays how the DNA falls alongside a molecular weight marker. This helps confirm the expected identity of the DNA you isolated. If so, take clear images of the gel and save copies for your notebook. 

Additional Tasks

A professional in the field of Laboratory Technician may also perform these tasks:

  • Prepping DNA- tissue type and cell types

  • Isolating/extracting dna from blood

  • Depts - Molecular and microbiology; biology; genomics and genetics; tissue procurement; neurology and neuroscience; cell biology; pathology and immunology (basic science depts); bioengineering

  • Nucleic acid quantification using Qbit

  • Nanodrop and purity readings 260/280 readings; reading protocols and understand readings

  • Sizing readings using Bioanalyzer and Tape station - is it fully entact and not fragmented or degraded; interpreting quality and quantity of DNA

  • Safety training

  • Lab culture - where and how is data stored (notebook, excel, databases, locations)

  • Cleanliness - keeping bench clean and non-contamination

  • Personal safety and sample safety

  • Hazardous materials - BSL2, hepatitis vaccine

  • Maintain lab records

  • Prepare solutions

  • Order lab supplies

Skills Used to Perform this Task

 General Skills

  • Ability to ask questions; if doing manually

  • Systematic, Repetitive, consistent mindset

  • Attention to detail

  • Cleanliness and organization

  • Responsible and self-starter

Biotechnology Skills:

  • Volume calculations for small dilutions

  • Proper use of the following equipment: micropipettes and small tubes, nanodrop, potentially gel electrophoresis chamber and gel imager)

  • Following documentation requirements

Skills Used in the Field

  • Confidence to ask questions when uncertain

  •  Presentation skills (putting data into a presentable format. Can be Microsoft Powerpoint, spreadsheets, or lab notebook, or written reports) 

Career Progression

Research technician I, II or Research lab technician I, II > Senior research technician

Sample Job Postings

Typical Pay Range: $16-25/hr

WashU

Research Laboratory Technician I | Pay rate: $16-22/hr

Research Laboratory Technician II | Pay rate: $16-25/hr

Danforth Plant Science Center

Laboratory Technician | Pay rate: $17-20/hr

Sample Job Posting: Research Laboratory Technician

Scheduled Hours 37.5

Position Summary Our lab studies regeneration in aquatic segmented worms, such as the marine annelid Platynereis dumerilii, and freshwater annelid Pristina leidyi.

The successful candidate will perform their independent project under the PIâ™s supervision, assist the PI with experiments and with performing general lab and animal culture maintenance tasks.

Job Description

Primary Duties & Responsibilities
• Help PI with microinjections.
• Carry out confocal imaging for experiments and injected samples.
• Carry out image analysis and prepare figures.
• Help maintain injected animal cultures (breeding, feeding, genotyping, fixing).
• Carry out other molecular biology tasks such as preparing in vitro mRNA, in situ hybridization experiments.
• Perform other duties as assigned.

Working Conditions

This position works in a laboratory environment with potential exposure to biological and chemical hazards. The individual must be physically able to wear protective equipment and to provide standard care to research animals.

Applicant Special Instructions


Documents to be included in the application:
• A cover letter explaining your career goals and why you want to work with us.
• A CV or academic resume.
• Unofficial transcripts.
• Contact information of three references.

Preferred Qualifications


• B.A./B.S. in biology, cell and molecular biology, developmental biology, or a related field.
• Molecular biology (e.g., PCR, genotyping, running DNA gels, preparing solutions). It is critical that the candidate is able to make calculations for preparing solutions, figuring out concentrations, etc.
• For any other skills required for the position, training will be provided by the lab members and/or by the PI.

Required Qualifications

High school diploma or equivalent high school certification.