1.

Solutions and Calculations

●​ Metric and Unit Conversions: While the sources don't provide a specific guide or
practice sheet for calculations, they extensively use metric units for volumes (µL, mL),
weights (mg, µg, ng), concentrations (ug/ml, ng/ml, µM), temperatures (°C), time
(minutes, hours), and speed (rpm, x g). Calculating the required volumes for reactions
based on desired final volumes and stock concentrations is a common task.
●​ Making Gels and Running Buffer: Agarose gels are used for separating DNA
fragments. A common concentration mentioned is 1% agarose gel. TAE buffer is used
for agarose gel electrophoresis. The concentration of ethidium bromide used for staining
is mentioned as 0.5 ug/ml

●​ Various Solutions and their Roles:

○​ LB Broth (Luria Broth): A nutrient-rich medium used for growing bacteria. It
contains growth factors and nutrients to help cells recover after transformation
and promote rapid division.
○​ Antibiotics (Ampicillin, Tetracycline): Used as selection factors on agar plates.
Ampicillin resistance gene (AmpR) is often present on plasmids. Plates
containing ampicillin allow only bacteria that have taken up the plasmid to grow,
selecting for transformants. Tetracycline is also mentioned in some plates.
○​ IPTG: Induces the expression of T7 RNA polymerase in the HT115(DE3) feeding
strain, which is necessary for producing double-stranded RNA (dsRNA) from the
cloned insert for RNA interference.
○​ Ethidium Bromide: A stain that binds to DNA and fluoresces under UV light,
allowing DNA bands to be visualized on a gel.
○​ Loading Dye (10x): Added to DNA samples before loading onto an agarose gel.
It typically contains a dense substance to help the sample sink into the well and
tracking dyes to monitor the progress of electrophoresis.
○​ Deionized Water (dH2O): Used as a solvent in many reactions and dilutions.
○​ Restriction Enzyme Buffer (10X): Provides the optimal conditions (salts, pH) for
restriction enzymes to efficiently cut DNA.
○​ Ligation Buffer (10X): Provides the necessary conditions for DNA ligase to form
phosphodiester bonds and join DNA fragments.
○​ CaCl2 Solution: Used in the chemical transformation protocol to make bacterial
cell membranes more permeable to DNA. It likely helps neutralize the negative
charge of the DNA and the cell membrane.
○​ LB Broth (post-heat shock): Added after heat shock during transformation to
help the stressed bacterial cells recover and stabilize their membranes before
plating.
○​ Spectrophotometry Buffers (e.g., dH2O, Elution Buffer): Used as a blank or
for diluting DNA samples before measuring absorbance.
○​ Plasmid Miniprep Kit Buffers (Monarch & PureLink):
■​ Buffer B1 (Resuspension Buffer): Resuspends bacterial cell pellets.
Contains Tris-HCl (buffering) and EDTA (metal chelator). RNase A is
added to this buffer to degrade RNA.
 ■​ Buffer B2 (Lysis Buffer): Lysates bacterial cells. Contains SDS
(detergent, dissolves membranes, denatures proteins) and NaOH
(denatures DNA). The solution becomes viscous.
■​ Buffer B3 (Neutralization Buffer): Neutralizes the lysis reaction.
Contains Potassium Acetate (KOAc) and likely other components like
Tris-Cl and EDTA. Adding this buffer causes a precipitate containing
chromosomal DNA, proteins, and lipids to form, separating them from the
plasmid DNA which remains in solution.
■​ Binding Buffers (B2, B3/HC for PCR Purification, L3 for Gel
Extraction): Used in conjunction with chaotropic salts to create
conditions where DNA binds selectively to the silica membrane in the spin
column. Gel Solubilization Buffer (L3) also helps dissolve the agarose gel
slice.
■​ Wash Buffers (BZ, WZ for Miniprep, W1 for PCR Purification & Gel
Extraction): Used to wash away residual contaminants from the silica
membrane. Contain ethanol to maintain DNA binding to the silica.
Removing residual ethanol is important before elution.
■​ Elution Buffers (EY, E5, E1, TE): Low salt buffers (e.g., 10 mM Tris-HCl,
pH 8.5 or TE buffer) or water (pH >7.0) used to release the purified DNA
from the silica membrane.
2. Restriction Enzymes and Cut Sites
●​ Function: Restriction enzymes are bacterial endonucleases that recognize and cleave
double-stranded DNA at specific sequences. They are crucial tools for cloning and
moving DNA fragments.
●​ Our Plasmid (L4440) and Digestion: The L4440 plasmid is used as the vector for
subcloning the target gene. It contains a Multiple Cloning Site (MCS) where the gene
of interest is inserted. The MCS is flanked by two T7 promoters running in opposite
directions. The plasmid also contains an Origin of Replication (Ori site) for replication
in bacteria, and an Ampicillin Resistance gene (AmpR) as a selectable marker.
●​ Enzymes Used: In this cloning project, XbaI and KpnI restriction enzymes are used to
digest both the L4440 plasmid and the purified PCR product (insert). Using the same
enzymes generates compatible sticky ends, allowing for directional or "forced" cloning.
●​ Expected Digestion Products:
○​ Digestion of the L4440 plasmid within the MCS with XbaI and KpnI releases a
small "stuffer fragment" (the original MCS sequence). This leaves the larger
linearized vector backbone.
○​ Digestion of the PCR product containing the gene of interest with XbaI and KpnI
generates a fragment with complementary sticky ends to the digested vector.
●​ Gel Analysis of Digests: After restriction digestion, samples are run on an agarose gel
to separate the fragments by size.
○​ The digested L4440 plasmid should appear as a single band corresponding to
the size of the linearized vector (~2.79 kb). Complete digestion is essential, as
undigested circular or partially digested forms can interfere with subsequent
steps like ligation and transformation.
 ○​ The digested PCR product (insert) should appear as a single band at its
expected size (~1.2-1.5 kb).
○​ The small stuffer fragment runs very quickly and may run off the gel.
●​ Incomplete vs. Complete Digestion: Complete digestion means all recognition sites
have been cut by the enzymes. Incomplete digestion results in extra bands on the gel
corresponding to DNA molecules that were only cut at some of the sites. When
analyzing a diagnostic digest, bands should ideally be present in equimolar quantities,
with band intensity decreasing as size decreases (assuming uniform staining). Lighter or
brighter than expected bands can indicate partial digests or multiple fragments of similar
size (doublets). Gel purification helps isolate the desired completely digested fragments.
●​ Reading Maps, Tables, and Gel Bands: Be able to interpret gel images to identify
bands corresponding to the vector and insert based on their expected sizes and a DNA
ladder (size standard). Understand how to use tables to set up digestion reactions with
correct volumes of reagents. Read plasmid maps to locate features like the MCS, Ori,
AmpR, and promoter sites. Understand that restriction sites within the MCS are chosen
to allow insertion of the gene of interest without disrupting essential plasmid functions
like replication or selection. Appropriate restriction enzymes are those that cut within the
MCS and flank the gene of interest in the PCR product.
●​ Genetic Mapping of Plasmids: While source material mentions restriction mapping as
a technique, it doesn't detail how to construct a genetic map from digestion data.
However, reading existing plasmid maps is covered.
3. RNAi
●​ Mechanism: RNA Interference (RNAi) is a biological process where double-stranded
RNA (dsRNA) triggers the silencing or degradation of complementary messenger RNA
(mRNA), thereby reducing or eliminating the expression of a target gene.
○​ In C. elegans, dsRNA complementary to the target gene is introduced into the
cell (in this lab, by feeding worms bacteria producing it).
○​ The dsRNA is cleaved into shorter fragments called small interfering RNAs
(siRNAs) by an enzyme called Dicer.
○​ siRNAs then associate with the RNA-Induced Silencing Complex (RISC).
○​ The RISC complex, guided by the siRNA, finds and binds to complementary
mRNA molecules of the target gene.
○​ The target mRNA is then either degraded or its translation is blocked, leading to
reduced expression (knockdown) of the target gene.
●​ Enzymes Involved: Key enzymes mentioned are Dicer (cleaves dsRNA) and
components of the RISC complex. The bacterial T7 RNA polymerase is crucial for
producing the dsRNA in the feeding strain.
●​ What is Happening: RNAi is used as a tool to study gene function by reducing gene
expression. The effect of the gene knockdown on the organism (phenotype) is observed
and compared to wild-type.
●​ Difference from Knocking out a Gene: This specific difference is not explicitly detailed
in the provided sources. However, based on the mechanism described, RNAi typically
results in a reduction of gene expression ("knockdown"), while gene knockout implies a
 complete elimination of gene function (the gene is permanently altered or removed from
the genome).
4. Model Organisms
●​ Definition: Model organisms are a few dozen species that have been extensively
studied because they are small, easy and fast to grow and manipulate in the
laboratory, and can be used to probe basic biological questions. Their genetic studies
often provide insights into biological processes.
●​ Organisms Discussed:
○​ C. elegans: A small (~1 mm adult), free-living roundworm. Its genome has been
sequenced, and it's widely used for RNAi studies. It is easy to manipulate and
has been extensively studied genetically. Phenotypes can be observed to infer
gene function.
○​ E. coli: A bacterium that is the most commonly used organism for molecular
cloning. It divides every 20-30 minutes, allowing for rapid growth and
amplification of plasmids. DNA (plasmids) can be easily introduced into E. coli by
transformation. Specific strains like DH5alpha are high-efficiency transformants,
while HT115(DE3) is specially modified for RNAi experiments by lacking RNase
III and having inducible T7 RNA polymerase.
5. Plasmid Isolation (Miniprep)
●​ Goal: To isolate and purify plasmid DNA from bacterial cells, separating it from
chromosomal DNA, RNA, proteins, and other cellular components. Enough plasmid is
needed for downstream applications like restriction digestion and transformation.
●​ Important Ideas and Steps:
○​ Bacterial Culture: Cells containing the plasmid are grown in liquid culture (LB +
antibiotic) to a sufficient density.
○​ Harvesting Cells: Bacterial cells are collected by centrifugation, forming a pellet.
The supernatant (liquid medium) is discarded.
○​ Resuspension: The cell pellet is resuspended in a buffer (e.g., Monarch Buffer
B1 or GTE buffer). This buffer contains Tris-HCl (buffering) and EDTA (chelates
divalent metal ions, which protects DNA from nucleases). RNase A is added to
this buffer to degrade bacterial RNA, as RNA would co-purify with the plasmid
DNA.
○​ Lysis: An alkaline lysis solution (e.g., Monarch Buffer B2 or SDS/NaOH) is
added. SDS (Sodium Dodecyl Sulfate) is a detergent that breaks down cellular
membranes (cell wall and plasma membrane), disrupting lipids and causing their
release. SDS also denatures proteins and can inactivate enzymes. NaOH is an
alkaline solution that denatures DNA (both plasmid and chromosomal). Due to
their supercoiled structure, plasmid DNA strands remain entangled after
denaturation.
○​ Neutralization: A neutralization buffer (e.g., Monarch Buffer B3) is added,
containing components like Potassium Acetate (KOAc). The high salt
concentration in the neutralization buffer allows the denatured chromosomal
DNA, which is much larger and tangled, to re-anneal incorrectly and aggregate,
forming a precipitate. The denatured proteins (bound to SDS) and lipids also
 precipitate. The smaller, supercoiled plasmid DNA, which renatures correctly,
remains in the soluble fraction (supernatant).
○​ Separation/Binding: The mixture is centrifuged to pellet the precipitate
containing chromosomal DNA, proteins, and debris. The supernatant, containing
the plasmid DNA, is transferred to a silica-based spin column. In the presence
of chaotropic salts (often present in binding buffers like B2, B3/HC, L3), the DNA
binds to the silica membrane.
○​ Washing: Wash buffers containing ethanol are passed through the column by
centrifugation to remove contaminants that are not bound to the silica. Residual
ethanol must be removed by a final centrifugation step to avoid interfering with
downstream enzymatic reactions like ligation.
○​ Elution: A low-salt buffer (Elution Buffer E5, E1, TE) or water (pH >7.0) is added
to the silica membrane. Under these conditions, the DNA is released from the
silica and collected by centrifugation into a clean tube.
I. Fundamental Concepts & Organisms
●​ Model Organisms: Small, easy and fast to grow and manipulate, used to study basic
biological questions, often genetically studied. Examples include E. coli, S. cerevisiae, C.
elegans.
●​ Molecular Biology Tools: Many advances are due to the ability to clone genes.
●​ Plasmids: Circular DNA molecules that replicate independently, used to make many
copies of DNA fragments. Plasmids often contain a selectable marker to allow
identification of cells containing the plasmid under specific conditions.
●​ Caenorhabditis elegans (C. elegans): A small (about 1 mm), free-living roundworm
used as a model organism. It is transparent and the first multicellular organism to have
its genome completely sequenced. C. elegans is usually grown with E. coli OP50 as a
food source.
II. DNA Separation and Visualization: Agarose Gel Electrophoresis
●​ Purpose: To slow down the movement of DNA and separate it by size. Allows
visualization and size determination of DNA fragments.
●​ Principle: DNA is negatively charged. When placed in an electrical field, DNA migrates
toward the positive pole (anode).
●​ Agarose Gel: A porous matrix made of polymerized agarose that allows DNA to move
through it. The density of the gel (agarose concentration) affects DNA migration speed
and the range of sizes that can be separated.
●​ Migration Speed: Depends on the size of the DNA, strength of the electrical field, buffer,
and gel density. Small DNA moves faster than large DNA.
●​ Equipment: Gel tank, power supply, casting tray, gel combs, electrical leads.
●​ Procedure Overview:
○​ Gel Preparation: Combine agarose powder and buffer solution, boil until clear,
cool slightly (~60°C), and pour into the casting tray with combs submerged. The
gel polymerizes upon cooling (30-45 minutes).
○​ Setup: Place the gel in the electrophoresis chamber. Add enough
electrophoresis buffer to cover the gel and fill the wells. Connect electrical leads
to the power supply (DNA migrates toward the red/positive anode).
 ○​ Sample Preparation: Mix DNA samples with 10X sample loading buffer. This
buffer contains Bromophenol Blue for color (tracking dye) and Glycerol for
weight (increases density so sample sinks into wells).
○​ Loading: Carefully pipette the mixed sample into the wells. Avoid puncturing the
gel. The sample should sink into the well.
○​ Running: Turn on the power supply. Bubbles should form on the electrodes. The
tracking dye (Bromophenol Blue) front also migrates through the gel.
●​ Visualization:
○​ Ethidium Bromide: A powerful mutagen. It binds to DNA and fluoresces
under UV light, allowing visualization of DNA bands.
○​ Can be added to the gel and/or running buffer before the run or the gel can be
stained after running.
○​ Staining involves placing the gel in warm diluted stain (25-30 mins), followed by
destaining in water to remove excess stain.
○​ Requires an ultraviolet light source to visualize DNA bands.
●​ DNA Ladder: Molecules of known size run alongside samples to determine the size of
restriction fragments.
III. DNA Purification
●​ Plasmid Purification (Miniprep):
○​ Purpose: To isolate plasmid DNA from bacterial cells.
○​ Method (Silica Gel based kits): Resuspend bacterial cell pellet in buffer (Buffer
B1). Lyse cells using lysis buffer (Buffer B2 - contains SDS). Neutralize to
precipitate chromosomal DNA and proteins (Buffer B3). Centrifuge to pellet
debris. Transfer supernatant (containing plasmid) to a silica spin column. Plasmid
DNA binds to the silica membrane. Wash the membrane to remove contaminants
(Buffer BZ, W1). Elute purified DNA from the column using elution buffer (Buffer
EY, E5, or water). Centrifugation is used throughout the process.
○​ Kit Used: Monarch® Spin Plasmid Miniprep Kit. PureLink® Silica Gel Plasmid
MiniPrep.
○​ Key Components/Buffers: Buffer B1 (Resuspension), Buffer B2 (Lysis, contains
SDS), Buffer B3 (Neutralization). Buffer BZ (Wash 1), Buffer WZ (Wash 2). Buffer
EY or E5 (Elution). RNase A (added to Buffer B1 to degrade RNA). Isopropanol
(added to Buffer BZ). Ethanol (added to Buffer WZ).
○​ Why it works: Takes advantage of the physical properties of plasmids (circular,
smaller than chromosomal DNA). Alkaline lysis denatures DNA, but smaller
circular plasmids can re-anneal, while larger chromosomal DNA aggregates and
precipitates. Silica membrane selectively binds dsDNA in the presence of
chaotropic salts.
●​ PCR Purification:
○​ Purpose: To efficiently remove primers, dNTPs, enzymes (like Taq polymerase),
and salts from PCR products.
○​ Method (Silica-based kit): Mix PCR product with Binding Buffer. dsDNA binds to
the silica-based membrane in a spin column. Wash column with Wash Buffer to
remove impurities. Elute dsDNA with low salt Elution Buffer or water.
 ○​ Kit Used: PureLink® PCR Purification Kit.
○​ Buffers: Binding Buffer (B2) for 100 bp-12 kb fragments, Binding Buffer
High-Cutoff (B3) for removing fragments <300 bp like primer dimers. Wash Buffer
(W1) with ethanol. Elution Buffer (E1 - 10 mM Tris-HCl, pH 8.5) or water.
○​ Importance of removing ethanol: Ethanol interferes with downstream
enzymatic activities like ligation and PCR.
●​ Gel Purification:
○​ Purpose: To separate a specific DNA fragment from a mixture on an agarose gel
and purify it from the agarose matrix for use in subcloning or other applications.
○​ Advantage: Allows working with a specific piece of DNA.
○​ Method (Kit-based): Electrophorese DNA on an agarose gel. Visualize with
ethidium bromide and UV light. Quickly excise the desired band from the gel
with minimal excess agarose to minimize UV exposure and maximize recovery.
Transfer gel slice to a tube. Weigh the gel slice (1 mg = 1 µL). Add Gel
Solubilization Buffer (L3) at a specific ratio to the gel weight (e.g., 3:1 for <2%
agarose). Incubate at 50°C to dissolve the gel. Load dissolved gel onto a silica
spin column. Centrifuge to bind DNA. Wash column with Wash Buffer (W1)
containing ethanol. Centrifuge to remove residual wash buffer/ethanol. Elute DNA
with Elution Buffer (E5) into a clean tube.
○​ Kit Used: PureLink® Quick Gel Extraction Kit.
○​ Buffers: Gel Solubilization Buffer (L3), Wash Buffer (W1), Elution Buffer (E5).
○​ Considerations: Choose appropriate gel concentration and electrophoresis
conditions for effective separation. Avoid overloading the gel.
IV. DNA Modification: Enzymatic Reactions
●​ Restriction Digestion:
○​ Purpose: To cut double-stranded DNA at specific recognition sequences using
restriction endonucleases.
○​ Restriction Enzymes: Bacterial enzymes that cleave DNA. They are supplied in
50% glycerol and must be kept on ice.
○​ Applications: Linearize plasmids, excise inserts, diagnostic digest.
○​ Diagnostic Digest: Confirms the presence of an expected insert in a plasmid by
cutting the plasmid with restriction enzymes and analyzing the resulting fragment
pattern on an agarose gel.
○​ Procedure: Calculate reagent volumes (DNA, 10X buffer, water, enzymes) for a
final volume (e.g., 25 µL or 30 µL). Add buffer, water, and DNA to tubes before
adding enzymes. Add enzymes last. Flick or tap to mix, then spin down briefly.
Incubate reaction (typically 1 hour at 37°C). Digests can be stored at -20°C.
○​ Materials: Plasmid/PCR product DNA, 10X Restriction Enzyme Buffer,
Restriction Enzymes, Deionized Water, Ice.
○​ Interfering Factors: Contaminants and methylation can interfere with enzyme
activity. Glycerol concentration should be less than 5% of the total reaction
volume.
○​ Complete Digest: Fragments should be in equimolar quantities; band intensity
on a gel should decrease with fragment size. Undigested vector (supercoiled
 form) transforms more effectively than linearized DNA, so complete digestion of
the vector is essential for successful cloning.
●​ Dephosphorylation:
○​ Purpose: To remove the phosphate groups from the 5' ends of DNA using an
enzyme like phosphatase.
○​ Enzyme: rSAP (recombinant Shrimp Alkaline Phosphatase).
○​ Application in Cloning: Dephosphorylating the linearized vector prevents it
from re-circularizing on itself, forcing the insert to ligate into the vector. The insert
is typically not dephosphorylated, as its 5' phosphate is needed for ligation to the
vector.
○​ Procedure: Incubate DNA with rSAP and buffer (CutSmart® Buffer) at 37°C for
30 minutes. Stop the reaction by heat-inactivation at 65°C for 5 minutes.
●​ Ligation:
○​ Purpose: To join a DNA fragment (insert) into a linearized vector using DNA
ligase. This process creates a phosphodiester bond between the DNA ends.
○​ Enzyme: T4 DNA ligase (a type of DNA ligase mentioned as relevant).
○​ Requirements: Requires a ligation buffer which creates the necessary
conditions for the DNA ligase to work.
○​ Reaction Setup: Uses purified, digested, and sometimes dephosphorylated DNA
fragments. An insert to vector ratio of 3:1 or 6:1 is often used, with an excess of
insert to increase the probability of it interacting with the vector.
○​ Incubation: Often incubated overnight at a reduced temperature (10-12°C).
○​ Why cold temperature: Slows down molecular movement, increasing the
chance of the insert and vector interacting and ligating successfully.
○​ Stuffer Fragment: The small fragment (like the multiple cloning site) removed
from the vector during digestion. It runs off the gel quickly due to its small size.
It's important to purify the linearized vector away from the stuffer fragment to
prevent it from ligating back into the vector instead of the desired insert.
V. Introducing DNA into Cells: Transformation
●​ Purpose: To introduce exogenous DNA (like a plasmid) into competent bacterial cells.
●​ Competent Cells: Bacterial cells that are capable of taking up external DNA. Not all
bacterial cells are naturally competent.
●​ Strains Used:
○​ NE5 / DH5 alpha: High efficiency transformants, healthy strain, divides quickly,
produces many copies of plasmid. Used to generate a high concentration of the
subcloned plasmid.
○​ HT115(DE3): The feeding strain for RNAi. It is RNase III deficient (to prevent
degradation of double-stranded RNA) and contains an IPTG-inducible T7 RNA
polymerase (to generate target gene dsRNA from the plasmid). This strain is less
healthy than DH5 alpha due to modifications to its chromosome (RNase gene
removed).
●​ Transformation Methods:
 ○​ Heat Shock: A method involving rapid temperature changes (cold to hot to cold)
that creates temporary pores or instability in the cell membrane, allowing DNA to
enter.
○​ Electroporation: Uses electricity to create instability and pores in the cell
membrane.
●​ Key Reagents:
○​ Calcium Chloride (CaCl2): Helps DNA associate with the negatively charged
bacterial cell surface, neutralizes the negative charge repulsion between DNA
and the cell, and pulls DNA close to the cell surface.
○​ LB Broth: Added after heat shock to help cells recover from the stress of
transformation. Provides nutrients and growth factors that trigger cells to stabilize
membranes and express genes for metabolism and reproduction.
●​ Procedure (Heat Shock): Resuspend cells in CaCl2 solution on ice. Add plasmid DNA
(or controls like dH2O or original vector) to the cell suspension on ice. Incubate on ice
(e.g., 15 minutes). Heat shock by transferring tubes from ice to a 42°C water bath for a
short time (e.g., 90 seconds). Immediately return tubes to ice. Add LB broth to the tubes
for recovery. Plate cell suspension onto selective media. Incubate plates (e.g., 37°C
overnight) to allow colonies to grow.
●​ Selection for Transformants: Plasmids often contain a selectable marker, such as
antibiotic resistance (e.g., Ampicillin). Plating transformed cells on agar plates containing
the antibiotic allows only cells that have successfully taken up the plasmid (and thus the
resistance gene) to grow and form colonies.
●​ Positive and Negative Controls (Transformation):
○​ Positive Control: Transformation with the original L4440 plasmid (without the
insert). Expected to grow on selective plates (e.g., LB/amp/Tet) as the plasmid
confers resistance.
○​ Negative Control: Transformation with water or no DNA. Expected to show no
growth on selective plates.
○​ Target Gene Sample: Transformation with the ligated plasmid containing the
insert. Expected to grow on selective plates if transformation was successful.
●​ Blue-White Screening: An alternative method to identify transformants containing the
insert. Uses plates with X-gal and relies on the disruption of the lacZ gene (encoding
β-galactosidase) in the vector's multiple cloning site by the inserted DNA. Colonies with
the insert (non-functional lacZ) are white, while those without the insert (functional lacZ)
are blue. The L4440 vector used in the labs is not suitable for blue-white screening as it
lacks the lacZ gene in its MCS.
VI. Bacterial Culturing and Plating
●​ Purpose: Grow bacterial cells containing plasmids to sufficient density for downstream
applications like plasmid purification. Also used to prepare bacterial lawns for feeding C.
elegans.
●​ Culturing: Can be done in liquid medium (like LB broth) in tubes/flasks or on solid
medium (agar plates). Liquid cultures are often incubated with shaking (e.g., 37°C at
190-225 rpm) to provide aeration and even growth.
 ●​ Agar Plates: Dilute suspension of cells spread on agar plates allows individual cells to
grow into individual colonies.
●​ Colony Forming Unit (CFU): Each colony theoretically arises from a single cell,
meaning all cells in a colony are genetically uniform.
●​ Picking Colonies: Selecting a well-isolated colony from an agar plate using a sterile
pipette tip.
●​ Starter Colony & Starter Culture: Picking a single colony (starter colony) and growing
it in liquid broth creates a starter culture, ensuring genetic uniformity for subsequent
experiments.
●​ Aseptic Technique: Using sterile procedures (e.g., sterile tips, keeping plates covered,
disinfecting) to prevent contamination from unwanted microorganisms.
●​ Spreading Cells: Spreading the cell suspension evenly over the agar surface using a
spreader to ensure isolated colonies.
●​ Media:
○​ LB (Luria Broth): A rich liquid medium for bacterial growth.
○​ LB Agar Plates: Solid medium for growing colonies.
○​ Selective Plates: LB agar containing antibiotics (e.g., Ampicillin, Tetracycline) to
select for bacteria containing the plasmid with the resistance gene.
○​ NGM Plates: Nematode Growth Medium plates used for growing C. elegans,
typically seeded with bacteria as a food source.
○​ NGM-Lite Plates + Ampicillin + Tetracycline and IPTG: Used for growing the
HT115(DE3) feeding strain containing the RNAi plasmid. Ampicillin and
Tetracycline select for bacteria with the plasmid, and IPTG induces the T7 RNA
polymerase to produce dsRNA.
●​ Feeding Strains for C. elegans:
○​ OP50: Wild-type E. coli used as a food source (negative control for RNAi). Uracil
auxotroph, resulting in a limited lawn on NGM plates, which is desirable for
observing and mating worms.
○​ HT115(DE3): E. coli strain used for RNA interference. Contains the RNAi plasmid
targeting a specific C. elegans gene.
●​ Seeding Plates: Applying bacterial culture (OP50 or HT115(DE3)) to the surface of agar
plates (NGM or NGM-Lite) to create a bacterial lawn for worms.
VII. DNA Quantification and Analysis: Spectrophotometry
●​ Purpose: To determine the concentration and purity of DNA samples. Often done
using a Nanodrop spectrophotometer.
●​ Principle: Measures the absorbance of light by the sample at specific wavelengths.
Nucleic acids (DNA and RNA) absorb UV light most strongly at 260nm, while proteins
absorb most strongly at 280nm.
●​ Measurements: Absorbance is measured at 260nm and 280nm.
●​ Purity Assessment: The ratio of absorbance at 260nm to 280nm (A260/A280 ratio) is
used to assess purity. A ratio of approximately 1.8-2.0 is considered pure DNA [Source
does not specify ideal ratio, this is general knowledge].
●​ Concentration Calculation: Absorbance at 260nm is used to calculate DNA
concentration (specific conversion factors are used).
VIII. DNA Sequencing: Sanger Sequencing
●​ Purpose: To determine the specific sequence of nucleotides in a DNA fragment. Used to
confirm the sequence of an insert in a plasmid.
●​ Principle: Based on the selective incorporation of chain-terminating
dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.
●​ ddNTPs: Dideoxynucleotide triphosphates lack a 3' hydroxyl (-OH) group. When a
ddNTP is incorporated into a growing DNA strand, it terminates chain extension because
there is no 3' OH to form a phosphodiester bond with the next incoming nucleotide.
●​ Procedure Overview (Cycle Sequencing): Uses a single-stranded DNA template, an
oligonucleotide primer, deoxynucleoside triphosphates (dNTPs), fluorescently labeled
ddNTPs, and a heat-stable DNA polymerase. Multiple cycles of denaturation, annealing,
and extension generate a series of DNA fragments of varying lengths, each ending with
a labeled ddNTP.
●​ Analysis: The fragments are separated by size using electrophoresis (with single-base
resolution). A detector reads the fluorescent label at the end of each fragment as it
passes through, allowing the sequence to be determined.
●​ Advantages (compared to some Next Generation Sequencing): Can produce longer
sequence reads (>500 nucleotides) and has a very low error rate (accuracies around
99.99%).
●​ Kit Used: BigDye Terminator v3.1 Cycle Sequencing Kit. Reagents should be protected
from light.
●​ Materials: Purified plasmid DNA with insert (template), primer, deionized water, BigDye
Terminator Ready Reaction Mix.
IX. Working with C. elegans in RNAi Experiments
●​ RNA Interference (RNAi): A method used to "knock down" the expression of a specific
gene. In these labs, it is induced by feeding worms bacteria engineered to produce
double-stranded RNA (dsRNA) corresponding to the target gene sequence.
●​ Target Gene Selection: Can be based on wild-type gene function or association with a
disease. Understanding the function of the C. elegans homolog of a gene is important.
●​ Feeding Strain: The HT115(DE3) E. coli strain containing the L4440 plasmid with the
target gene insert is used as the feeding strain. The plasmid drives expression of dsRNA
corresponding to the insert sequence.
●​ Seeding Plates for RNAi: Applying HT115(DE3) culture (with RNAi plasmid) or OP50
culture (negative control) to NGM-Lite plates containing appropriate supplements
(Ampicillin, Tetracycline, IPTG). Plates are allowed to grow for ~48 hours to establish the
bacterial lawn.
●​ Worm Transfer: Aseptic technique is crucial to prevent contamination. Worms are
transferred to seeded plates using a toothpick with a small bead of bacteria ("glue").
Requires gentle technique to avoid damaging the worms.
●​ Worm Development Stages: Identifying different larval stages (L1, L2, L3, L4) and
adults. The L4 stage is important for setting up crosses.
●​ Scoring Plates & Analyzing Data: Observing worms under a microscope on control
and RNAi plates. Comparing treated worms to wild-type (OP50-fed) and positive control
worms (fed bacteria targeting a gene with a known strong phenotype, e.g., dumpy).
 Scoring involves observing phenotype (size, movement, appearance,
mechanosensation) and counting the number of worms displaying a mutant phenotype
vs. wild-type. Gentle touch assay (using an eyebrow hair) can be used to test
mechanosensation defects. Calculating the percentage of worms with a particular
phenotype is important for analysis. RNAi is not always 100% penetrant.
X. Laboratory Workflow / Integrated Procedures (Typical Order)
Based on the descriptions of various procedures and the list of previous/future labs, a common
flow is:
1.​ Genomic DNA Prep (from C. elegans) and Plasmid Purification (of L4440 vector).
2.​ PCR Amplification of the target gene from genomic DNA. Includes positive and
negative controls.
3.​ PCR Purification to clean the amplified target gene product.
4.​ Restriction Digestion of the PCR product (insert) and the L4440 plasmid (vector) using
compatible enzymes (e.g., XbaI & KpnI). Enzymes must cut within the Multiple Cloning
Site (MCS) of the vector.
5.​ Agarose Gel Electrophoresis of the digested products to visualize and separate the
fragments.
6.​ Gel Purification of the desired digested insert and linearized vector bands from the gel.
The excised MCS ("stuffer fragment") is small and runs off the gel.
7.​ Dephosphorylation of the linearized vector to prevent re-circularization.
8.​ Ligation of the purified, digested, and dephosphorylated vector with the purified,
digested insert. Typically done overnight at cold temperatures.
9.​ Transformation of a high-efficiency E. coli strain (like DH5 alpha) with the ligation
product.
10.​Plate transformed bacteria on selective media (e.g., LB + Ampicillin).
11.​Pick Colonies from the plates and grow overnight cultures in selective liquid medium.
12.​Plasmid Purification (Miniprep) from selected colonies to isolate the plasmid DNA.
13.​Diagnostic Restriction Digest on the isolated plasmid DNA to confirm the presence
and correct size of the insert.
14.​Agarose Gel Electrophoresis of the diagnostic digest to analyze the fragment pattern.
15.​Sanger Sequencing (optional but recommended) to further validate the insert
sequence.
16.​Transformation of the HT115(DE3) feeding strain with the confirmed plasmid containing
the insert.
17.​Seed Plates (NGM-Lite with supplements) with the transformed HT115(DE3) strain
(target gene) and controls (OP50 and positive control feeding strain).
18.​Transfer Worms (C. elegans) to the seeded plates.
19.​Score Plates and analyze the RNAi phenotype data observed in the worms.
Primer Design (for Subcloning)
●​ Primers are short DNA sequences that bind to target DNA to amplify it using PCR.​

●​ Add restriction sites (e.g., KpnI and XbaI) to the 5' end of primers so enzymes can later
cut outside the gene without damaging it.​
 ●​ Important primer features:​

○​ Tm (melting temp): ~60°C is ideal.​

○​ Forward and reverse primers should have similar Tm values.​

○​ GC content should be ~50%.​

○​ GC clamp: end primers with G or C at 5’ end for better binding.​

○​ Avoid primer dimers (primers binding to each other).​

○​ Avoid hairpins and other secondary structures.

Restriction Enzymes (REs)
●​ Bacterial enzymes that cut DNA at specific sites.​

●​ KpnI and XbaI used to cut both the PCR product and vector (L4440).​

●​ Creates sticky ends for easier and more efficient ligation.​

●​ The vector is linearized and the stuffer fragment (middle part) is removed.​

●​ DNA fragments are run on a gel, visualized with UV, and purified.
Transformation (Introducing Plasmids into Bacteria)
●​ Transfers plasmid DNA into bacteria, like E. coli.​

●​ Uses competent cells made via CaCl₂ treatment.​

●​ Heat shock method: DNA + cells on ice → 42°C → ice → recovery in LB broth.​

●​ Plated on selective media (e.g., with ampicillin) to grow transformed cells.​

●​ Use DH5α strain for cloning; use HT115(DE3) for RNAi feeding.​

●​ HT115(DE3) lacks RNase III and has T7 RNA polymerase, allowing RNAi plasmid
expression.
Controls in Experiments
Transformation Controls:
●​ Vector + Insert (Ligation 1): Should grow if ligation/transformation worked.​

●​ Vector Only (Ligation 2): Should have little/no growth if digestion/dephosphorylation

worked.​
 ●​ Positive Control (uncut L4440): Should show growth (tests transformation success).​

●​ Negative Control (no DNA): Should have no colonies (tests contamination/selectivity).​

PCR Controls:
●​ Positive Control (known DNA): Should amplify expected band (tests PCR reagents).​

●​ Negative Control (no DNA): Should have no band (tests for contamination).​

RNAi Worm Plate Controls:

●​ OP50 Control: Normal worms, no RNAi, shows wild-type phenotype.​

●​ Positive Control (e.g., Dumpy RNAi): Confirms RNAi system is working.​

●​ Target Gene RNAi: Compares phenotype to OP50 to assess gene function.​

RNAi in C. elegans
●​ Worms eat HT115(DE3) bacteria making dsRNA for your gene.​

●​ dsRNA is processed into siRNAs, which guide RISC to degrade the gene’s mRNA.​

●​ This reduces protein expression, letting you observe phenotype changes.​

●​ Common phenotypes: dumpy, uncoordinated, roller, long.​

●​ RNAi is not 100% penetrant — not all worms will show effects, and effects may vary.​