Big Lab Flowchart by

Transfer "transformation solution" into two tubes

saanvigupta10 — 2024-06-06 12:58:26 UTC

The solution helps prepare the cells for transformation by making them able to take up plasmid DNA

Place the tubes on ice

saanvigupta10 — 2024-06-06 12:59:17 UTC

Increases the cell membrane permeability to the plasmid DNA

Suspend bacterial colonies in "transformation solution"

saanvigupta10 — 2024-06-06 13:02:00 UTC

Gets the cells ready to take up plasmid DNA

Add pGLO plasmid DNA to the "+pGLO" tube

saanvigupta10 — 2024-06-06 13:03:05 UTC

Introduces the plasmid to the cells

BACTERIAL TRANSFORMATION

saanvigupta10 — 2024-06-06 13:03:55 UTC

Incubate the tubes on ice

saanvigupta10 — 2024-06-06 13:09:40 UTC

Helps plasmid DNA bind to cell membranes

Heat-shock and then place is back on ice

saanvigupta10 — 2024-06-06 13:11:56 UTC

The heat opens the pores of the cells and allows the plasmid to enter, and then the cooling closes the pores, preventing it from escaping the cell

Add LB broth and incubate at room temperature

saanvigupta10 — 2024-06-06 13:17:18 UTC

Allows the cells to recover and express the antibiotic resistance gene

Plate cells on agar plates and incubate

saanvigupta10 — 2024-06-06 13:18:10 UTC

Allows bacterial colonies to grow and express the plasmid

INOCULATION

saanvigupta10 — 2024-06-06 13:21:27 UTC

Examine plates with UV light

saanvigupta10 — 2024-06-06 13:30:39 UTC

Checks for GFP expression in the transformed colonies

Inoculate 2 tubes, one labeled "+" and the other "-"

saanvigupta10 — 2024-06-06 13:32:15 UTC

Prepares cultures for protein production

Cap the tubes and shake vigorously

saanvigupta10 — 2024-06-06 13:35:23 UTC

Ensures even distribution of bacteria in the tubes

Incubate for 2 days, then at room temperature

saanvigupta10 — 2024-06-06 13:36:52 UTC

Promotes bacterial growth and GFP production

PROTEIN PURIFICATION

saanvigupta10 — 2024-06-06 13:38:26 UTC

A. Bacterial Concentration

saanvigupta10 — 2024-06-06 13:39:25 UTC

Transfer 2mL of culture into a microtube and centrifuge

saanvigupta10 — 2024-06-06 13:41:55 UTC

Pellets the bacterial cells

Pour out supernatant, leaving only the pellet

saanvigupta10 — 2024-06-06 13:43:35 UTC

Isolates the bacterial cells for lysis

Add the solution to the tube and resuspend the pellet

saanvigupta10 — 2024-06-06 13:46:10 UTC

Prepares for bacterial lysis

Add a drop of lysozyme and mix gently, then freeze

saanvigupta10 — 2024-06-06 13:49:11 UTC

Begins lysing the bacterial cells and preserves sample for the next experiment section

B. Bacterial Lysis

saanvigupta10 — 2024-06-06 13:51:03 UTC

Thaw the microtube and centrifuge

saanvigupta10 — 2024-06-06 13:53:25 UTC

Separates the lysate from the cell debris

Prepare the HIC chromatography column

saanvigupta10 — 2024-06-06 13:56:05 UTC

Sets up for protein purification

Add aliquots of the supernatant into the top of the column

saanvigupta10 — 2024-06-06 13:59:43 UTC

Loads the lysate onto the column for separation

Centrifuge

saanvigupta10 — 2024-06-06 14:00:38 UTC

Helps the binding of proteins to the column

Transfer binding solution to another microtube

saanvigupta10 — 2024-06-06 14:03:02 UTC

Prepares for protein extraction and storage

C. Protein Chromatography

saanvigupta10 — 2024-06-06 14:05:58 UTC

Remove the caps of the column, place in a tube, and let it drip until in reaches the surface of the HIC matrix

saanvigupta10 — 2024-06-06 14:07:00 UTC

Begins the protein separation process

Load the supernatant into the top of the column

saanvigupta10 — 2024-06-06 14:08:27 UTC

Separates GFP from other proteins

Add wash buffer and let the solution pass through the column

saanvigupta10 — 2024-06-06 14:11:15 UTC

Washed away unbound proteins

Add TE buffer and let the entire volume flow through the column

saanvigupta10 — 2024-06-06 14:12:14 UTC

Removes GFP protein from the column

Transfer column to tube just before the GFP drips, and then wrap the tube and refrigerate

saanvigupta10 — 2024-06-06 14:13:14 UTC

Collects purified GFP

PROTEIN ELECTROPHORESIS

saanvigupta10 — 2024-06-06 14:20:00 UTC

Get the protein samples and prepare them for electrophoresis

saanvigupta10 — 2024-06-07 11:28:26 UTC

Analyzes the purity and the size of the GFP protein

Thaw the samples

saanvigupta10 — 2024-06-07 11:29:04 UTC

Prepares them for loading onto the gel

Add 15 microliters of appropriate loading buffer into each new tube

saanvigupta10 — 2024-06-07 11:30:35 UTC

Increases density of sample and provides visible markers

Heat the samples for 5 minutes

saanvigupta10 — 2024-06-07 11:31:27 UTC

Denatures the proteins for separation

Place tubes in heat block

saanvigupta10 — 2024-06-07 11:32:49 UTC

Ensures the proteins have fully denatured

Load samples into the wells of the gel

saanvigupta10 — 2024-06-07 11:34:11 UTC

Prepares for protein separation by electrophoresis

Place the top on the chamber and connect to power supply

saanvigupta10 — 2024-06-07 11:34:58 UTC

Begins electrophoresis to separate protein by size

Run the gel for 35 minutes

saanvigupta10 — 2024-06-07 11:35:40 UTC

Separates the proteins by size

Stain the gel to visualize the proteins

saanvigupta10 — 2024-06-07 11:36:06 UTC

Analyzes the protein bands