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RNA Concentration Calculator

🧮 Nanodrop → Concentration

A₂₆₀ reading

Dilution factor

Sample type

A₂₆₀/A₂₈₀

A₂₆₀/A₂₃₀

Please enter a valid A₂₆₀ value.

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RNA Molarity Calculator

🧮 Concentration → Molarity

molarity (M) = conc (g/L) / (length (nt) × 340 g/mol/nt)

Average MW per nucleotide ≈ 340 g/mol (ssRNA). Each nt contributes ~330 Da + ~10 Da for phosphate backbone.

RNA concentration

Conc unit

Template length (nt)

Avg MW/nt (g/mol)

Please enter concentration and template length.

Molarity

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Quantification & Quality Control

📊 Nanodrop / Spectrophotometry

conc (µg/µL) = A₂₆₀ × 40 × dilution factor

Ratio	Acceptable	Interpretation
A₂₆₀/A₂₈₀	1.8 – 2.1	<1.8 = protein/phenol contamination; >2.1 = possible RNA degradation
A₂₆₀/A₂₃₀	2.0 – 2.2	<1.8 = chaotropic salt, phenol, or guanidinium contamination

Extinction coefficients:
ssRNA: 40 µg/µL per A₂₆₀ unit · ssDNA: 33 · dsDNA: 50
Always blank with the same buffer used to resuspend RNA.

🔬 Bioanalyzer / Gel QC

Metric	Notes
RIN score	RNA Integrity Number 1–10. ≥8 for RNA-seq. ≥7 acceptable for RT-qPCR.
28S:18S ratio	~2:1 for intact total RNA on gel/Bioanalyzer.
Gel appearance	Two sharp rRNA bands (28S, 18S). Smearing indicates degradation.
mRNA	Broad smear ~0.5–5 kb for poly-A selected mRNA — this is normal.
Ladder	Always run an RNA ladder (not DNA). Use formaldehyde gels for total RNA.

Storage Conditions

🧊 RNA Storage Guidelines

Condition	Duration / Notes
On ice (4°C)	Short-term only (<1h). Use for active work.
–20°C	Weeks. Store in nuclease-free H₂O or 10 mM Tris pH 7.0, 1 mM EDTA. Avoid repeated freeze-thaw.
–80°C	Months to years. Preferred for long-term storage. Aliquot to avoid freeze-thaw cycles.
Ethanol ppt.	Store as EtOH precipitate at –80°C for very long-term. Pellet, don't resuspend until needed.
Aliquots	Make single-use aliquots of precious RNA. Thaw on ice, use once, discard.
Resuspension	Resuspend in nuclease-free H₂O or TE (10 mM Tris pH 7.0, 1 mM EDTA). Avoid pH >8 (RNA hydrolysis).

RNA Handling — Critical Rules

⚠️

RNase contamination is the #1 cause of RNA degradation. Wear fresh gloves at all times. Change gloves after touching skin, door handles, or shared equipment. Never pipette without gloves.

🧤 Workspace & Contamination Control

Rule	Detail
Gloves	Wear nitrile gloves; change frequently. RNases from skin are highly stable.
Dedicated area	Use an RNA-only bench or hood. Do not bring DNA work materials in.
Surfaces	Wipe bench with RNaseZap or 70% EtOH + RNase decontamination spray before starting.
Tubes & tips	Use certified RNase-free (individually wrapped) consumables. Do not reuse.
Equipment	Dedicated pipettes for RNA work. Treat with RNaseZap periodically.
Ice	Keep RNA on ice at all times unless performing a timed step.

💧 RNase-free Water & Reagents

Reagent	Preparation / Notes
DEPC-H₂O	Add 0.1% DEPC, incubate 12h at 37°C, autoclave to inactivate DEPC. Use for all RNA buffers.
Commercially RNase-free H₂O	Preferred for most applications — no DEPC residue risk. Nuclease-free certified.
NaOH (1M)	Naturally RNase-free; can be used to treat solutions that cannot be autoclaved.
Tris buffers	Must be prepared with DEPC-H₂O or nuclease-free H₂O. Cannot use DEPC directly — it reacts with amines.
EDTA	Chelates Mg²⁺ required by RNases — include 1 mM EDTA in storage buffers to inhibit RNase activity.

Common RNA Protocols

📋 TRIzol / Total RNA Extraction (key steps)

Step	Detail
Homogenise	Lyse in 1 mL TRIzol per 50–100 mg tissue or per 5–10×10⁶ cells. Pipette vigorously.
Phase sep.	Add 0.2 mL chloroform. Shake 15s, incubate 3 min RT, centrifuge 12,000×g 15 min 4°C.
Aqueous phase	Transfer upper aqueous phase (~600 µL). Do not disturb interface.
Precipitation	Add 0.5 mL isopropanol. Mix, incubate 10 min RT, centrifuge 12,000×g 10 min 4°C.
Wash	Wash pellet with 1 mL 75% EtOH (DEPC-H₂O). Vortex, centrifuge 7,500×g 5 min 4°C.
Dry & resuspend	Air-dry 5–10 min (not over-dry). Resuspend in 20–50 µL nuclease-free H₂O, 55°C 10 min.

🔄 RT-qPCR — Key Considerations

Step	Notes
DNase treatment	Always treat RNA with DNase I before RT to remove genomic DNA contamination.
RT input	Use consistent input mass (e.g. 500 ng or 1 µg). Normalise across samples.
No-RT control	Include –RT control per sample to detect gDNA contamination.
Primer design	Span an exon-exon junction to prevent gDNA amplification.
Reference genes	Use ≥2 stable reference genes (e.g. GAPDH + ACTB). Validate stability.
Efficiency	Primer efficiency should be 90–110% (slope –3.1 to –3.6).
Melt curve	Always run a melt curve to confirm single product.

⚠️ Common RNA Mistakes

Mistake	Consequence / Fix
Touching face → tube	RNase contamination. Change gloves immediately.
Using non-DEPC H₂O	RNase activity degrades RNA. Always use certified nuclease-free H₂O.
Over-drying pellet	RNA becomes insoluble. Air dry max 10 min; pellet should be slightly translucent.
Repeated freeze-thaw	RNA degradation. Aliquot and use single-use aliquots.
High pH resuspension	RNA hydrolyses at pH >8. Resuspend in pH 7.0 Tris or nuclease-free H₂O.
Skipping –RT control	gDNA amplification will be mistaken for mRNA signal.
Vortexing RNA	Mechanical shearing of large RNA. Mix by gentle pipetting or flicking instead.

Passage Calculator

🧮 Seeding & Passage Calculator

cells to seed = target density × vessel area

Current cell count (×10⁶)

Viability (%)

Target vessel

Area (cm²)

Seeding density (×10⁴/cm²)

Media volume (mL)

Please fill in cell count and area.

Cells to seed

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Common Human Cell Lines

🔬 HeLa & HEK293 Reference

Cell line	Details
HeLa	Cervical adenocarcinoma. Epithelial morphology. Doubling time ~24h. Split 1:5–1:10 every 2–3 days.
HEK293	Human embryonic kidney. Epithelial. Doubling ~36h. Very transfectable. Split 1:4–1:8 every 2–3 days.
HEK293T	HEK293 + SV40 T antigen. Ideal for transient transfection & lentiviral production. Split 1:5–1:10.
HEK293FT	Optimised for high-titre lentivirus production. Split 1:5–1:20.

📐 Seeding Densities

Vessel	Area	Media	HeLa seed	HEK293 seed
96-well	0.32 cm²	0.1–0.2 mL	5–10k	10–20k
48-well	0.95 cm²	0.5 mL	15–25k	25–50k
24-well	2 cm²	1 mL	30–50k	50–100k
12-well	3.5 cm²	2 mL	50–100k	100–200k
6-well	9.5 cm²	3–4 mL	150–250k	300–500k
T25	9.6 cm²	5–7 mL	200–300k	300–500k
T75	25 cm²	12–15 mL	500k–1M	1–1.5M
T175	75 cm²	30–40 mL	1.5–3M	3–5M

Media & Reagents

🧪 Standard Media Formulations

Cell line	Media	Supplements
HeLa	DMEM high glucose	10% FBS, 1% Pen/Strep, 1% GlutaMAX
HEK293	DMEM high glucose	10% FBS, 1% Pen/Strep
HEK293T	DMEM high glucose	10% FBS, 1% Pen/Strep, 1 mM Na-pyruvate
HEK293FT	DMEM high glucose	10% FBS, 1% NEAA, 1% Pen/Strep, 500 µg/mL G418 (optional)

📋 Reagent Reference

Reagent	Working conc.	Notes
FBS	10% v/v	Heat-inactivate 30 min at 56°C. Aliquot and store –20°C.
Pen/Strep	1% (100 U/mL)	100× stock. Omit for transfection if possible.
GlutaMAX	1% (2 mM)	Stable L-glutamine substitute. Preferred over L-Gln.
L-Glutamine	2 mM	Unstable — degrades at 4°C. Replace every 2–3 weeks or use GlutaMAX.
Trypsin-EDTA	0.05–0.25%	37°C, 2–5 min. Neutralise with serum-containing media. Don't over-trypsinise.
DMSO (cryo)	10% v/v	In 90% FBS. Add DMSO last, on ice. Slow-freeze –1°C/min to –80°C, then LN₂.
PBS	1× pH 7.4	For washes. Must be Ca²⁺/Mg²⁺-free for detachment steps.

Critical Rules

⚠️ Sterile Technique & Contamination

Rule	Detail
Hood	Spray down with 70% EtOH, UV 15 min before use. Work with sash at correct height.
Gloves	Spray with 70% EtOH before entering hood. Never touch face or non-sterile surfaces mid-work.
Media warming	Warm media to 37°C before use. Cold media shocks cells.
Mycoplasma	Test monthly. Common source: FBS, shared reagents, other cell lines. No visible sign until severe.
Cross-contamination	Work one cell line at a time. Use separate reagent aliquots per line.
Passage number	Record and track. High-passage cells drift phenotypically. Thaw new stock every 10–15 passages.
Incubator	37°C, 5% CO₂, 95% humidity. Check water tray weekly. Clean monthly.

🧊 Freezing & Thawing

Step	Detail
Freeze density	1–3×10⁶ cells/mL per vial in freezing medium (90% FBS + 10% DMSO).
Slow freeze	Mr Frosty or isopropanol cryo-container at –80°C overnight, then transfer to LN₂.
Thawing	37°C water bath ~1 min. Transfer to 9× volume warm media immediately to dilute DMSO.
Post-thaw	Spin 300×g 3 min, remove DMSO-containing supernatant, resuspend in fresh media.
DMSO toxicity	DMSO is cytotoxic at RT — work fast, keep on ice, remove promptly after thawing.
First passage	Check viability 24h post-thaw before splitting. Cells need time to recover.

💡 Troubleshooting

Problem	Likely cause / fix
Cells not attaching	Cold media, old trypsin, flask not tissue-culture treated, or poor viability post-thaw.
Slow growth	Low passage FBS lot issue, CO₂/pH off, glutamine depleted, mycoplasma.
Media turns yellow fast	Cells too confluent or CO₂ imbalance. Check incubator CO₂ level and cap tightness.
Floating cells	Over-trypsinised, over-confluent, or contamination. Check under microscope.
Low transfection efficiency	Check confluency (60–80% ideal), DNA quality, reagent ratio. Avoid Pen/Strep.
Mycoplasma suspected	Quarantine immediately. Test with PCR or DAPI staining. Treat or discard and thaw clean stock.

Solid → Solution

⚖️ How Much to Weigh Out

mass (g) = conc (M) × MW (g/mol) × volume (L)

Quick-pick reagent (optional)

MW (g/mol)

Purity / hydrate factor

Target concentration

Conc unit

Final volume

Volume unit

Please fill in MW, concentration, and volume.

Weigh out

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Protocol

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C₁V₁ = C₂V₂ — Liquid Dilution

🧪 Dilute from Stock

V₁ = (C₂ × V₂) / C₁

Stock conc (C₁)

C₁ unit

Final conc (C₂)

C₂ unit

Final volume (V₂)

Volume unit

Check inputs — final concentration must be less than stock.

Stock to add (V₁)

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% w/v

💧 % w/v Solution

g = (% × volume_mL) / 100

% w/v

Volume (mL)

Please fill in both fields.

Mass to dissolve

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Mass → Molarity

⚗️ Calculate Molarity

M = (mass_mg / 1000) / MW / volume_L

Mass (mg)

MW (g/mol)

Volume

Volume unit

Please fill in all fields.

Molarity

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Molarity → Mass

⚖️ Mass to Weigh Out (simple)

mass (g) = M × MW × volume_L

Target (M)

MW (g/mol)

Volume

Volume unit

Please fill in all fields.

Mass to weigh

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Henderson–Hasselbalch

🔬 pH / Buffer Ratio Calculator

pH = pKa + log([A⁻] / [HA])

Buffer system

pKa

Target pH

Total [buffer] (mM)

Volume (mL)

0246781014

Check inputs. pH should be within ±2 of pKa for effective buffering.

Buffer components

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Common Buffers

📋 Buffer Reference

Buffer	pH Range	pKa
Acetate	3.6 – 5.6	4.76
MES	5.5 – 6.7	6.10
MOPS	6.5 – 7.9	7.02
Phosphate	5.8 – 8.0	7.20
HEPES	6.8 – 8.2	7.55
Tris-HCl	7.0 – 9.0	8.06
Bicine	7.6 – 9.0	8.35
Glycine	8.6 – 10.6	9.60

💡 Buffer Tips

Note	Detail
Tris temp.	pKa drops ~0.03/°C — calibrate pH at use temperature
EDTA	Will not dissolve below pH 7 — adjust to pH 8.0 while stirring
Buffering range	Effective within pKa ± 1 pH unit
Autoclave	Do not autoclave phosphate + Mg²⁺/Ca²⁺ — precipitates
RNA buffers	Prepare all RNA buffers in DEPC-H₂O or nuclease-free H₂O
Tris + RNA	Cannot use DEPC to treat Tris — DEPC reacts with amines. Use pre-made nuclease-free Tris.

Molecular Weights

📦 Common Reagents

Compound	MW (g/mol)	Tag
NaCl	58.44	salt
KCl	74.55	salt
MgCl₂·6H₂O	203.30	hydrate
CaCl₂·2H₂O	147.01	hydrate
Na₂HPO₄	141.96	buffer
NaH₂PO₄	119.98	buffer
EDTA (free acid)	292.24
EDTA·Na₂·2H₂O	372.24	hydrate
Tris base	121.14	buffer
Tris·HCl	157.60	buffer
HEPES	238.31	buffer
Glucose	180.16
Sucrose	342.30
SDS	288.38	detergent
DTT	154.25	reducing
β-mercaptoethanol	78.13	reducing

Quick Formulas

📐 Equations

Formula	Expression
Molarity	M = n / V(L)
Dilution	C₁V₁ = C₂V₂
% w/v	g / 100 mL
% v/v	mL / 100 mL
Henderson-H.	pH = pKa + log([A⁻]/[HA])
RNA conc	µg/µL = A₂₆₀ × 40 × dilution
Beer-Lambert	A = ε × c × l

RNA Lab Reference · Mol Bio / Biochem