Technical Specification: DNA Purification & Gel Recovery Kits¶

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1. Product Overview¶

Product	SKU	Application	Elution Volume	Binding Capacity
Agarose Gel DNA Recovery Kit	D1200	Purify DNA from TAE/TBE agarose gels	≥30 μL	>15 μg per column
DNA Purification Kit	D1300	Clean up PCR products, enzymatic reactions, restriction digests	≥30 μL	>10 μg per column

1.1 Key Advantages¶

Advantage	D1200 (Gel Recovery)	D1300 (PCR Cleanup)
Gel dissolving time (1% TAE, 200 mg slice)	5–10 min at 56°C	N/A (solution cleanup)
Volume handling	≤ 400 μL dissolved gel	≤ 100 μL sample input
Fragment retention	100 bp – 10 kb	100 bp – 10 kb
Downstream compatible	Ligation, restriction, sequencing, labeling	Ligation, restriction, sequencing, labeling
Protocol time	15–20 min	10–15 min
No phenol/chloroform	Yes	Yes
Residual agarose	< 0.1% in eluate	N/A

2. Technical Parameters¶

2.1 Gel DNA Recovery Kit (D1200)¶

Parameter	Specification
Technology	Silica membrane spin column
Fragment size range	100 bp – 10 kb
Recovery efficiency (>80%)	100 bp – 8 kb
Recovery efficiency (30–50%)	8 kb – 10 kb
Gel buffer compatibility	TAE, TBE
Binding capacity	>15 μg per column
Elution volume	30–50 μL (minimum 25 μL for maximum concentration)
Gel dissolving buffer pH	5.0–6.0 (containing NaI or guanidine thiocyanate)

2.2 DNA Purification Kit (D1300)¶

Parameter	Specification
Technology	Silica membrane spin column
Fragment size range	100 bp – 10 kb
Recovery efficiency	>80% (100 bp – 8 kb)
Input volume	≤100 μL standard; up to 500 μL with multiple loading
Binding capacity	>10 μg per column
Elution volume	30–50 μL
Compatible samples	PCR products, restriction digests, labeling reactions, kinase reactions, ligation reactions
Residual primer removal	>95% removal of primers < 40 nt
Residual dNTP removal	>99% removal

2.3 Recovery Efficiency by Fragment Size¶

Fragment Size	D1200 (Gel Recovery)	D1300 (PCR Cleanup)
100–200 bp	70–85%	75–90%
200–500 bp	80–95%	85–95%
500 bp – 3 kb	85–95%	85–95%
3–5 kb	80–90%	80–90%
5–8 kb	70–85%	70–85%
8–10 kb	30–50%	40–60%
> 10 kb	10–30%	15–35%

2.4 Binding Principle¶

DNA fragments selectively adsorb to the silica membrane in the presence of high concentrations of chaotropic salts (NaI in gel dissolving buffer, guanidine HCl in binding buffer). The chaotropic salt concentration required for effective binding to silica is:

[ [\text{Chaotrope}] \geq 3\text{ M for effective DNA binding} ]

At pH ≤ 7.5, DNA phosphate groups are protonated and interact with silanol groups on the membrane surface via hydrogen bonding and hydrophobic interactions. For gel extraction, the agarose is dissolved at 56°C in NaI-containing buffer, which simultaneously melts the agarose (gelling temperature 36–39°C) and provides binding conditions.

Elution is performed with low-ionic-strength buffer (10 mM Tris-HCl, pH 8.5). The alkaline pH deprotonates the silanol groups, increasing electrostatic repulsion, while the low salt concentration disrupts the chaotrope-mediated adsorption:

[ \text{DNA}{\text{(bound)}} \xrightarrow{\text{pH 8.5, low salt}} \text{DNA} ]}

3. Quality Control¶

Test	D1200	D1300
Endonuclease	Not detectable	Not detectable
Exonuclease	Not detectable	Not detectable
DNase/RNase	Not detectable	Not detectable
Residual ethanol	<0.1% in eluate	<0.1% in eluate
PCR inhibition	None at 5 μL eluate (50 μL PCR)	None at 5 μL eluate (50 μL PCR)
Ligation compatibility	>80% T4 ligation efficiency vs. column-free control	>80% T4 ligation efficiency vs. column-free control
Restriction digestion	Complete digestion of 1 μg eluted DNA	Complete digestion of 1 μg eluted DNA

4. Protocol Optimization¶

4.1 Optimization for Different Fragment Sizes¶

Fragment Range	Recommended Adjustments
100–200 bp	Use 30 μL elution buffer; incubate 5 min at RT before final spin; avoid overdrying membrane
200–500 bp	Standard protocol (50 μL elution)
500 bp – 3 kb	Standard protocol
3–8 kb	Use 30 μL pre-warmed elution buffer (56°C); avoid vortexing to prevent shearing
> 8 kb	Use wide-bore pipette tips; elute with 20 μL pre-warmed (56°C) EB; extend incubation to 5 min

4.2 Effect of Agarose Percentage on Recovery¶

Agarose %	Gel Slice Dissolving Time (min at 56°C)	Expected Recovery (500 bp)
0.7%	3–5 min	90–95%
1.0%	5–7 min	85–92%
1.5%	7–10 min	80–88%
2.0%	10–15 min	70–80%
3.0%	15–20 min (increase dissolving buffer to 4× gel volume)	50–65%

4.3 Ethanol Removal in Wash Step¶

Dry Spin Time at 12,000×g	Residual Ethanol in Eluate
1 min	0.5–1.0% (may inhibit downstream ligation)
2 min	< 0.1% (recommended)
3 min	< 0.05%
5 min	< 0.01%

5. Troubleshooting¶

Issue	Cause	Solution
Low recovery (< 50%)	DNA fragment too small (< 100 bp)	Add 1 volume isopropanol instead of ethanol; increase binding time to 5 min
	DNA fragment too large (> 10 kb)	Use pre-warmed elution buffer (56°C); avoid overdrying membrane; minimize pipetting shear
	Gel slice too large (> 400 mg)	Split into two columns or increase dissolving buffer to 4× gel volume
	Incomplete gel dissolution	Increase incubation time at 56°C to 15 min; vortex every 3 min
	Ethanol added incorrectly	Verify ethanol concentration (96–100%); do not use denatured ethanol with additives
No DNA recovered	Column over-dried	Do not exceed 2 min dry spin; overdrying reduces binding capacity
	Elution buffer wrong	Use EB provided (10 mM Tris-HCl, pH 8.5); do not use water (pH < 7)
	Ethanol precipitation caused DNA loss	Precipitated DNA may appear at tube bottom before column loading — ensure resuspension
DNA does not digest/ligate	Residual ethanol in eluate	Increase dry spin to 3 min; air-dry column 2 min at RT before elution
	Contaminating agarose/chaotropes	Reduce gel slice to ≤ 200 mg; increase wash steps to 3×
	Elution buffer incompatible	Dialyze or ethanol precipitate if using heat-sensitive downstream applications
A₂₆₀/A₂₈₀ < 1.7	Protein contamination	Increase wash volumes; add additional wash step
A₂₆₀/A₂₃₀ < 1.5	Chaotrope carryover	Increase wash; include a second wash with 80% ethanol added

6. Kit Components¶

D1200-100T¶

Component	Volume	Storage
Gel Dissolving Buffer	100 mL	RT
Wash Buffer (concentrate)	15 mL × 2	RT
Elution Buffer	30 mL	RT
Spin Columns	100 pcs	RT
Collection Tubes	100 pcs	RT

D1300-100T¶

Component	Volume	Storage
Binding Buffer	60 mL	RT
Wash Buffer (concentrate)	15 mL × 2	RT
Elution Buffer	30 mL	RT
Spin Columns	100 pcs	RT
Collection Tubes	100 pcs	RT

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