Reagents

Buffer P1 (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/ml RNase A); Lab 2 plasmid DNA isolation

Resuspends bacterial pellet. RNase A degrades RNA after lysis; EDTA chelates cations needed by DNases.

Buffer P2 (1% SDS, 0.2 M NaOH); Lab 2 plasmid DNA isolation

Lyses cells. SDS disrupts membranes/hydrophobic interactions; NaOH denatures proteins and nucleic acids.

Buffer N3 (3.0 M Potassium Acetate, pH 5.5); Lab 2 plasmid DNA isolation

Neutralizes lysate. Chromosomal DNA/protein precipitate (can't renature); small supercoiled plasmids renature correctly and stay in solution.

QIAprep spin column (silica membrane); Lab 2 plasmid DNA isolation

Selectively binds plasmid DNA under high-salt conditions.

Buffer PB; Lab 2 plasmid DNA isolation

Removes endonucleases to prevent DNA degradation.

Buffer EB (10 mM Tris-HCl, pH 8.5); Lab 2 plasmid DNA isolation

Low-salt elution buffer; releases DNA from silica membrane.

Sterile water; Lab 2 PCR

Brings reaction to final volume

5X Q5 DNA Polymerase Buffer; Lab 2 PCR

Optimal salt/pH/ionic conditions for polymerase.

dNTPs; Lab 2 PCR, Lab 6: Reverse Transcription & PCR

Nucleotide building blocks for new strand synthesis. Building blocks for cDNA synthesis.

Forward primer; Lab 2 PCR

Defines start of target region; anneals to one template strand.

Reverse primer; Lab 2 PCR

Defines end of target region; anneals to opposite strand.

DNA template (pBluescript II SK+); Lab 2 PCR

Source of the AmpR gene to be amplified.

Q5® High-Fidelity DNA Polymerase; Lab 2 PCR

Proofreading, heat-stable enzyme; produces blunt-ended PCR products.

Undigested pET24a MaSp1 4x vector; Lab 3 Restriction Digestion

Plasmid substrate to be cut.

10X rCutSmart Buffer; Lab 3 Restriction Digestion, Lab 4 PCR Product Digestion

Correct salt/pH for restriction enzyme activity.

ClaI; Lab 3 Restriction Digestion, Lab 4 PCR Product Digestion

Sticky-end cutter; cuts 5'-ATCGAT-3'. Blocked by Dam/Dcm methylation.

PsiI (PsiI-v2); Lab 3 Restriction Digestion, Lab 4 PCR Product Digestion

Blunt-end cutter; cuts 5'-TTATAA-3'

Agarose (1%); Lab 3 Agarose Gel Electrophoresis

Forms porous gel matrix/molecular sieve for size-based DNA separation.

0.5X TBE buffer (Tris, boric acid, EDTA); Lab 3 Agarose Gel Electrophoresis

Conducts current; Tris buffers pH, boric acid supplies ions, EDTA chelates DNase cofactors.

Ethidium bromide; Lab 3 Agarose Gel Electrophoresis

Intercalates DNA base pairs; fluoresces under UV for visualization. (Carcinogen — use gloves.)

1.0 kb DNA ladder; Lab 3 Agarose Gel Electrophoresis

Known-size markers for estimating fragment sizes.

10X Loading dye (EDTA, glycerol, tracking dye e.g. bromophenol blue); Lab 3 Agarose Gel Electrophoresis

EDTA inactivates DNases; glycerol sinks sample into well; tracking dye shows run progress.

Buffer QG (yellow; guanidine thiocyanate); Lab 3 Gel Purification (QIAquick-type)

Dissolves agarose; favors DNA-silica binding; pH ~6.6.

Silica spin column; Lab 3 Gel Purification (QIAquick-type)

Binds DNA from dissolved gel solution.

Buffer PE; Lab 2 plasmid DNA isolation, Lab 3 Gel Purification (QIAquick-type)

Ethanol-based wash; removes salts, keeps DNA bound.

Elution buffer (pH 8.5, low salt); Lab 3 Gel Purification (QIAquick-type)

Releases DNA from silica by disrupting binding conditions.

Gel-extracted PCR product; Lab 4 PCR Product Digestion

Insert (AmpR gene), blunt on both ends prior to digestion.

Heat (65°C, 15 min); Lab 4 PCR Product Digestion

Inactivates restriction enzymes before ligation.

10X Ligase Buffer (+ATP); Lab 4 Ligation

Provides salt/pH conditions; ATP is the energy source DNA ligase requires.

Digested vector DNA (pET24a MaSp1 4x); Lab 4 Ligation

Backbone with one sticky end, one blunt end.

Digested PCR product (AmpR gene); Lab 4 Ligation

Insert with matching sticky/blunt ends.

DNA Ligase; Lab 4 Ligation

Catalyzes phosphodiester bond formation, sealing insert into vector.

Ligation reaction; Lab 4 Transformation

Contains recombinant plasmid (pET24a MaSp1 AmpR).

Chemically competent E. coli (JM109); Lab 4 Transformation

Ca²⁺-treated bacteria; neutralizes charge repulsion so DNA can approach membrane.

Recovery media (no antibiotic); Lab 4 Transformation

Lets cells express AmpR before antibiotic selection.

LB agar + ampicillin plates; Lab 4 Transformation

Selects only for transformed (AmpR-expressing) colonies.

IPTG; Lab 5: Affinity Purification of His-Tagged Protein

Lactose analog; binds Lac repressor, induces T7 RNA polymerase → drives expression from T7 promoter.

LB broth + ampicillin; Lab 5: Affinity Purification of His-Tagged Protein

Growth/selection medium for expression strain.

BL21 DE3 pLysS cells (IPTG-induced); Lab 5: Affinity Purification of His-Tagged Protein

Expression strain; inactive RNaseE and lon/OmpT proteases protect mRNA/protein from degradation.

FastBreak™ Cell Lysis Reagent; Lab 5: Affinity Purification of His-Tagged Protein

Proprietary detergent/buffer cocktail; gently lyses cells, releasing native-state protein.

DNase I; Lab 5: Affinity Purification of His-Tagged Protein

Degrades DNA released during lysis, reducing viscosity.

MagneHis™ Ni-particles; Lab 5: Affinity Purification of His-Tagged Protein

Nickel-charged magnetic beads; 6xHis tag binds nickel, capturing tagged protein.

Magnetic stand; Lab 5: Affinity Purification of His-Tagged Protein

Pulls Ni-particles (with bound protein) out of solution for wash/elution steps.

Binding/Wash buffer; Lab 5: Affinity Purification of His-Tagged Protein

Removes unbound bacterial proteins while target stays bound.

Elution Buffer (500 mM imidazole); Lab 5: Affinity Purification of His-Tagged Protein

Imidazole competes with 6xHis tag for nickel binding, displacing/eluting purified protein.

1X DNA/RNA Protection Reagent; Lab 6: RNA Isolation

Stabilizes RNA; inactivates RNases upon resuspension.

Sonication; Lab 6: RNA Isolation

Physically disrupts cells to release contents.

RNA Lysis Buffer; Lab 6: RNA Isolation

Further lyses cells/denatures proteins (incl. RNases).

gDNA removal column; Lab 6: RNA Isolation

Removes contaminating genomic DNA before RNA binds.

Ethanol (95%); Lab 6: RNA Isolation

Adjusts conditions so RNA binds the purification column's silica membrane.

RNA purification column (silica); Lab 6: RNA Isolation

Selectively binds RNA.

RNA Wash Buffer; Lab 6: RNA Isolation

Removes contaminants; keeps RNA bound.

DNase I + Reaction Buffer; Lab 6: RNA Isolation

On-column digestion of residual genomic DNA.

RNA Priming Buffer; Lab 6: RNA Isolation

Washes away DNase I/buffer after digestion.

Nuclease-free water; Lab 6: RNA Isolation & Reverse Transcription

Elutes purified RNA without RNase contamination. Adjusts volume.

Total RNA (heat-denatured); Lab 6: Reverse Transcription

Template; heating removes secondary structure.

Oligo dT primer (23-mer); Lab 6: Reverse Transcription

Anneals to poly-A tail of mRNA; primes first-strand cDNA synthesis.

10X M-MuLV Buffer; Lab 6: Reverse Transcription

Optimal conditions for reverse transcriptase.

M-MuLV Reverse Transcriptase; Lab 6: Reverse Transcription

Synthesizes cDNA from mRNA template.

Water; Lab 6: PCR

Adjusts volume.

10X PCR buffer; Lab 6: PCR

Optimal conditions for Q5 polymerase.

RT reaction product (cDNA); Lab 6: PCR

Template for PCR.

Forward primer (CspA, w/ SalI site + RBS); Lab 6: PCR

Defines amplification start; adds cloning/expression elements.

Reverse primer (CspA, w/ XhoI, 6xHis tag, stop codon); Lab 6: PCR

Defines amplification end; adds tag/cloning elements.

Q5 DNA Polymerase; Lab 6: PCR

High-fidelity amplification of the CspA cDNA.

Purified His-tagged protein / positive control; Lab 7: Sample Prep & Gel

Samples analyzed.

Protein Loading Buffer: SDS; Lab 7: Sample Prep & Gel

Coats protein with negative charge proportional to mass; disrupts hydrophobic interactions.

Protein Loading Buffer: DTT or BME; Lab 7: Sample Prep & Gel

Breaks disulfide bonds (reduces tertiary/quaternary structure).

Protein Loading Buffer: pH 6.8 buffer; Lab 7: Sample Prep & Gel

Matches stacking gel pH for "ion sandwich" formation.

Protein Loading Buffer: Bromophenol blue; Lab 7: Sample Prep & Gel

Tracks electrophoresis progress.

Heat (95°C, 3 min); Lab 7: Sample Prep & Gel

Accelerates denaturation.

Polyacrylamide gel (stacking 4% / resolving 10–15%); Lab 7: Sample Prep & Gel

Fine mesh matrix; stacking concentrates proteins into a tight band, resolving gel separates by mass.

1X Running Buffer (Tris pH 8.3, glycine, 0.1% SDS); Lab 7: Sample Prep & Gel

Conducts current; glycine/chloride create stacking "ion sandwich"; SDS maintains charge coating.

Pre-stained MW marker; Lab 7: Sample Prep & Gel

Known-size protein standards for size estimation.

Nitrocellulose membrane; Lab 7: Western Transfer

Binds proteins via hydrophobic/H-bonding interactions; immobilizes proteins post-transfer.

1X Transfer Buffer (reduced/no SDS, 20% methanol); Lab 7: Western Transfer

Conducts transfer current; methanol improves protein-membrane binding.

Filter paper/fiber pads; Lab 7: Western Transfer

Structural sandwich components for even current flow.

Blocking buffer; Lab 7: Western Transfer

Occupies non-specific binding sites, prepping membrane for antibody probing.

Blocking solution (5% powdered milk w/v); Lab 8: Western Blot Immunodetection

Coats non-specific sites on membrane, preventing background antibody binding.

1X TBS-Tween (TBS-T); Lab 8: Western Blot Immunodetection

Wash/dilution buffer; Tween-20 detergent reduces non-specific binding.

Mouse anti-HIS primary antibody (1:500); Lab 8: Western Blot Immunodetection

Binds specifically to the 6xHis tag on the target protein.

Goat anti-mouse secondary antibody (1:5000, HRP-conjugated); Lab 8: Western Blot Immunodetection

Binds constant region of mouse antibody; carries HRP enzyme for signal generation.

SuperSignal West Pico Plus Luminol/Enhancer; Lab 8: Western Blot Immunodetection

Chemiluminescent substrate for HRP; produces light upon oxidation.

SuperSignal West Pico Plus Stable Peroxide; Lab 8: Western Blot Immunodetection

Hydrogen peroxide co-substrate for HRP reaction.

BioRad Chemiluminescence documentation system; Lab 8: Western Blot Immunodetection

Instrument; captures light signal as a visible band.

Unknown protein sample; Lab 9: In-Solution Tryptic Digestion

Target for identification.

Water; Lab 9: In-Solution Tryptic Digestion

Diluent.

6M Urea/100 mM Tris (pH 8); Lab 9: In-Solution Tryptic Digestion

Denatures protein by breaking H-bonds/hydrophobic forces (final 3M urea).

DTT (200 mM); Lab 9: In-Solution Tryptic Digestion

Reduces disulfide bonds.

Iodoacetamide (IAA, 200 mM); Lab 9: In-Solution Tryptic Digestion

Alkylates cysteine thiols, preventing disulfide re-formation.

DTT (100 mM, quench); Lab 9: In-Solution Tryptic Digestion

Neutralizes leftover unreacted IAA.

Trypsin (1 mg/ml); Lab 9: In-Solution Tryptic Digestion

Cleaves peptide bonds C-terminal to Lys (K)/Arg (R).

Trifluoroacetic acid (TFA, 10%); Lab 9: In-Solution Tryptic Digestion

Acidifies sample; improves peptide binding to C18 resin.

Activation Solution (50% ACN); Lab 9: C18 Spin Column (desalting/concentration)

Wets hydrophobic C18 resin.

Equilibration/Wash Buffer (0.5% TFA/5% ACN); Lab 9: C18 Spin Column (desalting/concentration)

Sets binding-compatible conditions on column. Removes salts/urea/DTT while peptides stay bound.

Elution Buffer (70% Acetonitrile); Lab 9: C18 Spin Column (desalting/concentration)

High-organic solvent disrupts hydrophobic binding, eluting purified peptides.