Week 1 Lab: Pipetting

Note: This document is a theoretical completion of the lab assignment.
I did not perform the experiments in person or virtually.
The answers below are based on pre‑lab reading, known formulas, and expected outcomes – provided solely to have the assignment completed.

Overview & Objective

This lab introduces foundational techniques of pipetting and serial dilutions. By the end, students should be able to use P20, P200, and P1000 pipettes accurately, perform dilutions, and prepare solutions with desired concentrations. The lab includes colour mixing (Part 1) and a serial dilution to obtain 100 µM of a mystery substance (MS) followed by a final reaction mix (Part 2).

Pre‑Lab Answers

Key Definitions (understood)

• Mole (mol): 6.022 × 10²³ particles.
• Molarity (M): moles of solute per litre of solution (mol/L).
• Conversions:
  1 L = 1000 mL = 1,000,000 µL
  1 M = 1000 mM = 1,000,000 µM

Dilution Practice 1

Goal: Dilute 5 M MS → 100 µM (0.1 mM) using sequential 1:499 and 1:99 steps.

• Step 1: 5 M = 5,000,000 µM.
  Dilute to 10,000 µM → 500‑fold dilution.
  1:499 means 1 part stock + 499 parts diluent (total 500 parts).
  Example: Take 10 µL of 5 M stock + 4990 µL (4.99 mL) dH₂O → 5000 µL of 10,000 µM.

• Step 2: 10,000 µM → 100 µM → 100‑fold dilution.
  1:99 means 1 part of the 10,000 µM solution + 99 parts diluent.
  Example: Take 10 µL of 10,000 µM + 990 µL dH₂O → 1000 µL of 100 µM.

Total dilution factor = 500 × 100 = 50,000, which correctly converts 5,000,000 µM to 100 µM.

Dilution Practice 2

Stock concentration in g/mL
Molar mass MS = 532 g/mol, concentration = 5 M.
5 mol/L × 532 g/mol = 2660 g/L = 2.66 g/mL (since 1 L = 1000 mL, 2660 g/L = 2.66 g/mL).

Plan to obtain 100 µM from 5 M
Total dilution needed: 5 M / 100 µM = 5 / 0.0001 = 50,000‑fold.
One easy 2‑step serial dilution:

1. 1:100 dilution – Prepare 50 mM (50,000 µM) from 5 M.
   Use P20: add 10 µL of 5 M stock + 990 µL dH₂O (using P1000) → 1 mL total in an Eppendorf tube. Mix well.
2. 1:500 dilution – From 50 mM to 100 µM (50,000 µM → 100 µM = 500‑fold).
   Take 10 µL of the 50 mM solution + 4990 µL dH₂O (use P1000 for water) → 5 mL total. Alternatively, do a smaller volume: 2 µL + 998 µL (using P20 and P1000).
   Final concentration = 100 µM.

Number of dilution steps: 2 steps (1:100 then 1:500).
Tubes: 1.5 mL Eppendorf tubes for intermediate steps; final tube can be a 5 mL or 15 mL tube if using larger volumes, or a PCR tube for small volumes.
Pipettes:

• P20 for 2–10 µL volumes.
• P200 for 20–200 µL (if needed).
• P1000 for 500–1000 µL additions.

Why make 100 µM MS when we need 40 µM?
Because 100 µM is a convenient intermediate concentration obtained from serial dilution. We then dilute that 100 µM stock to 40 µM in the final reaction mix (see table below). Preparing 40 µM directly from 5 M would require a single 125,000‑fold dilution, which is impractical (very tiny volumes, large error). Serial dilutions allow accurate, stepwise reduction.

Pre‑lab Table (Final reaction – 60 µL total)

Calculations:

• Loading dye: (C₁V₁ = C₂V₂) → 6X * V₁ = 1X * 60 µL → V₁ = 10 µL.
• MS: 100 µM * V₁ = 40 µM * 60 µL → V₁ = 24 µL.
• dH₂O: 60 – (10 + 24) = 26 µL.

Protocol Simulation (Theoretical – no physical or virtual lab performed)

Part 1 – Mixing Colors (expected steps)

• Tube 1, 2, 3: 500 µL each of red, yellow, blue (pure colours).
• Tube 4: 220 µL red + 220 µL yellow → orange.
  Add 200 µL first, then 20 µL (using P200, change tip between colours).
• Tube 5: 525 µL yellow + 525 µL blue → green.
• Tube 6: 155 µL red + 155 µL blue → purple.
• Plating design (if done): Spot 1, 2, 5, 10 µL on a Petri plate to visualise relative drop sizes.

Part 2 – Serial Dilution (to 100 µM MS) – theoretical procedure

1. First dilution (1:100):

   • In an Eppendorf tube, add 990 µL dH₂O (P1000).
   • Add 10 µL of 5 M MS (P20). Pipette up/down 3‑4 times.
   • Label: 50 mM (or 50,000 µM).

2. Second dilution (1:500):

   • In a new tube, add 4990 µL dH₂O (use P1000: 4 × 1000 µL + 990 µL).
   • Add 10 µL of the 50 mM solution (P20). Mix thoroughly.
   • Label: 100 µM MS.

3. Final reaction (60 µL total, from pre‑lab table):

   • In a PCR tube, add in order:
     • 26 µL dH₂O (P200)
     • 10 µL loading dye (P20)
     • 24 µL of the 100 µM MS (P200)
   • Mix by pipetting up/down.

4. Bonus – Gel loading (theoretical):

   • Take 20 µL from the final reaction.
   • Insert tip gently into a pre‑cast gel well (avoid puncturing the well bottom).
   • Expel slowly and withdraw tip vertically.

Expected Observations (if the lab had been performed)

• Colour mixing yields predictable secondary colours (orange, green, purple).
• Serial dilution visibly reduces colour intensity (if MS is dyed).
• Final reaction with loading dye appears purple (loading dye colour dominates).
• Gel loading shows sample settling into the well without spillage.