Molecular Biology Techniques: DNA Extraction, Cloning, and Analysis

Proteinase K

digests proteins

Buffer ATL

contains SDS (lyses cells, denatures proteins) and EDTA (chelates Mg/Ca, inhibits DNases)

Buffer AL

lysis buffer with guanidinium chloride; promotes DNA binding to silica

Buffer AW1 and AW2

wash buffers; keep DNA bound to column while contaminants are washed away

Elution buffer

releases DNA from the column into solution

EcoRI

cuts DNA at its recognition site, producing sticky ends

HindIII

cuts DNA at its recognition site, producing sticky ends

PCR components

primers, DNA template, Taq polymerase, dNTPs, buffer, water

pGEM-T easy vector

has T overhangs for ligation with PCR products, ampicillin resistance for selection, and an MCS inside lacZ

Ampicillin

selects for cells that took up the plasmid (only resistant cells grow)

X-gal

substrate for β-galactosidase; produces blue pigment when lacZ intact

IPTG

induces lac operon, enabling β-gal expression

LB

provides nutrients (growth medium)

Buffer P1

contains Tris-Cl (pH buffer) and RNase A (digests RNA)

Buffer P2

contains NaOH (denatures DNA) and SDS (lyses membranes)

Buffer N3

contains potassium acetate; neutralizes DNA, precipitates genomic DNA/proteins

Buffer PB

wash buffer 1; removes DNases, contains isopropanol + salts to keep plasmid DNA bound to column

Buffer PE

wash buffer 2; removes salts/ions, contains ethanol

Buffer EB

low-salt buffer that elutes plasmid DNA from the column

Beer's Law

absorbance (A) is directly proportional to concentration (c), expressed as A = kc

DNA absorbance wavelength

260 nm (due to nitrogenous bases)

Standard curve axes

x-axis = DNA concentration (ng/µl), y-axis = absorbance

Dilution formula

M1V1 = M2V2

Dilution example

to dilute 956 ng/µl DNA to 30 ng/µl in 500 µl → add 15.7 µl stock + 484.3 µl water

Restriction enzyme purpose

cut DNA at specific recognition sites

Sticky ends

complementary overhangs that allow ligation of insert and vector cut with same enzyme

Methylation

protects bacterial DNA from restriction enzyme cleavage

Gel electrophoresis

separates DNA by size and charge, DNA migrates negative → positive electrode

Smaller DNA fragments

migrate faster through gel

Genomic DNA after digest

appears as smear due to large size and many fragments

Lambda DNA after digest

distinct bands (smaller, defined pieces)

SYBR Green

stains DNA to visualize fragments under UV light

Molecular marker (DNA ladder)

provides reference fragment sizes for comparison

Circular (supercoiled) DNA migration

moves faster than linear DNA of same size

PCR purpose

amplify specific DNA sequence (e.g., insulin gene)

PCR cycle - Denaturation

95°C, separates DNA strands

PCR cycle - Annealing

55°C, primers bind to DNA template

PCR cycle - Extension

72°C, Taq polymerase synthesizes new DNA strand

PCR amplification equation

2ⁿ (n = number of cycles)

PCR enzyme activity

Taq polymerase adds extra A at 3′ ends of PCR product

Ligation purpose

insert PCR product into vector (pGEM-T easy)

Transformation purpose

introduce recombinant plasmid into E. coli cells

Heat shock transformation

42°C for 45 seconds opens pores, followed by ice to close them

pGEM-T easy vector features

T overhangs for PCR product, ampicillin resistance, MCS in lacZ gene

Blue colony

lacZ intact, β-gal digests X-gal → blue pigment, no insert present

White colony

lacZ disrupted by insert, β-gal inactive, X-gal not digested, insert present

Ampicillin in plates

selects for cells containing plasmid

IPTG in plates

induces lac operon, allowing β-gal expression

X-gal in plates

substrate that produces blue pigment if β-gal active

Miniprep purpose

isolate plasmid DNA from bacterial cells

EcoRI digestion results

blue plasmid (empty, ~3 kb), white plasmid (insert, ~4 kb)

Undigested plasmid migration

supercoiled plasmid migrates faster on gel