Purpose

The purpose of this introductory lab session is to familiarize participants with the lab safety practices and considerations for our work in the Biotechnology Program (BIT). These activities will help you identify lab eye wash stations, biohazard containers, and the different pipettors we will use.

Skills

This lab session will help you practice the following skills:

• Reading and carefully following a protocol.
• Correctly choosing and donning personal protective equipment (PPE).
• Critical thinking and experimental interpretation.
• Documenting the procedure followed and results obtained.

Knowledge

This lab session will help you gain the following knowledge:

• Familiarity with the general steps for accurate and reproducible pipetting.
• Selecting and properly using the correct pipettor for a given volume.
• Factors affecting pipetting accuracy.

Tasks

Review the lab safety video and discuss with your instructor concerns and questions.

Read the protocol for the miniPCR pipetting practice cards

We will spend time discussing the steps before demonstrating the setup of a Rapid Sequencing Kit (RSK) library prep and sequencing run.

The goal is to practice pipetting and learn from the demonstration. In future sessions, you will use frozen samples and total DNA to learn about the microbes and wax worm genes that could have caused the passing of Squeaky. To achieve this goal, you will work with your team to catalog, quantify, and prepare the DNA for sequencing using Oxford Nanopore Technologies (ONT). Let’s begin practicing!

Procedure

1. Review the Lab Safety video (9:41 min).
2. All materials will be considered biohazard and discarded in the biohazard waste bags to practice.
3. Clean your bench and pipettors with ethanol and a paper towel.
4. Review the procedure for the miniPCR practice cards and locate the four microfuge tubes with food coloring (red, green, blue, and yellow).
5. Identify the P10, P20, P200, and P1000 pipettes with gray, yellow, yellow, and blue button indicators.
6. Identify tips for the P10, P20, P200, and P1000 pipettes. Note that we will be using barrier or filter tips to minimize contamination.
7. Watch the miniPCR tutorial (5:40 min).
8. Review the first few minutes of the miniPCR demo using our Eppendorf pipettes (6:44).
9. Complete the miniPCR Pipetting tutorial and challenge. Record your observations on the handout.
10. (Optional) take a photograph of your colorful results.
11. Observe the Rapid Sequencing Kit library preparation demonstration.
12. Review the key steps listed below.
    1. Rapid Sequencing Kit V14 – gDNA (SQK-RAD114)-minion (PDF, 5 min)
13. Observe the flow cell priming and loading procedures for MinION flow cells.
14. Clean your bench and pipettors with ethanol and tidy up your workplace.

Criteria for Success

Pipetting is a critical skill for molecular biologists. We have the opportunity to practice with food coloring and your peers. Explore the pipettes available and their volume limitations. Completion of the miniPCR practice card activity is our goal. While successful pipetting may look different depending on your level of comfort and experience, practicing is our main objective.

Check Your Knowledge and Practice!