Ethachinmate

"Ethachinmate" is a high–molecular-weight carrier used for the precipitation of nucleic acids (DNA and RNA) with ethanol or isopropanol. It enables the efficient recovery of trace amounts of nucleic acids. It has been tested to be DNase-free and RNase-free.

Feature

・Recovery of trace nucleic acids

Enables quantitative recovery of DNA (>100 base pairs) and RNA (>120 bases) at concentrations above 20 ng/mL.

・Rapid alcohol precipitation

No incubation at –20 °C or –80 °C is required. Centrifugation can be performed immediately after the addition of alcohol.

・No inhibition of enzymatic reactions

Ethachinmate itself forms a visible pellet upon alcohol precipitation, reducing the risk of losing valuable nucleic acids, even in trace amounts, during washing.

・Visible precipitation

Once the ethanol is added, Ethachinmate itself forms a visible pellet. The risk of losing the pellet by washing is therefore reduced.

・Tested DNase and RNase-free

It has been confirmed to be DNase and RNase-free.

DNA or RNA solution (100 µl)
↓←3.3 µl, 3 mol/l Sodium Acetate(Product Component) ^*1
↓←1 µl, Ethachinmate ^*2
↓vortex ^*3
↓←200 - 250 µl, Ethanol
↓vortex ^*3
↓12 K x g for 5min at room temperature ^*4
pellet ^*5

• ^*1 Final salt concentration must be more than 0.1 mol/l.
• ^*2 Add 1 µl of Ethachinmate per 100 µl of DNA solution. If the amount of DNA solution is less than 100 µl, add 1 µl of Ethachinmate. If the amount of DNA solution is more than 300 µl, 3 µl of Ethachinmate is the correct amount to be added. Once Ethachinmate is added into DNA solution, there is no need to add Etachinmate again if performing more centrifugations. Adding to much Ethachinmate may make the solution viscous, causing the following processes to be difficult.
• ^*3 Vortexing improves the efficiency to recover subtle DNA.
• ^*4 Cooling is not necessary.
• ^*5 Pellet is visible. Pellet may then be dissolved in buffer and used as a template for enzyme reactions. Wash with 70 % ethanol, if needed.

RNA extraction protocol using ISOGEN with Ethachinmate

When total RNA is extracted using ISOGEN, the RNA pellet ma sometimes be difficult to see.
By adding Ethachinmate, the pellet is easier to see.

Sample
↓←ISOGEN
↓Homogenize and incubate at room temperature for 5min.
↓←Chloroform
↓Shake vigorously and incubate at room temperature for 3min.
↓Centrifuge for 15 min to separate the phases
↓Transfer the aqueous phase to a new tube
↓←1-2 µl of Ethachinmate ^*1
↓vortex
↓←Isopropanol.
↓Mix by gentle inversion and incubate at room temperature for 10 min.
↓Centrifuge for 10 min.
↓Remove the supernatant.
Pellet
↓←70 % Ethanol
↓vortex
↓Centrifuge for 5 min.
↓Remove the supernatant
Pellet
↓Air-dry the pellet.
↓←RNase-free water or TE buffer (pH 8.0) for dissolving the pellet.
Total RNA Solution

Recovery of Trace Amounts of Nucleic Acid

λ/HindIII fragments (10 ng in 500 µL) were precipitated using 1 mL of ethanol in the presence of 0.1 mol/L sodium acetate and analyzed by agarose gel electrophoresis.

Lane 1 : λ/HindIII (Contorol; no precipitation treatment)
Lane 2 : λ/HindIII+Ethachinmate 3 µl (immediate centrifugation)
Lane 3 : λ/HindIII Incubation at -20 °C overnight
Lane 4 : λ/HindIII Incubation at -80 °C for 20 min.

Result

Under the condition of 0.1 mol/L sodium acetate, adding Ethachinmate allows for quantitative recovery of trace amounts of DNA without the need for low-temperature incubation.

Low-Molecular-Weight RNA Ethanol Precipitation

Synthetic 21mer RNA samples (10 ng, 20 ng, 40 ng, and 80 ng) were subjected to ethanol precipitation under the following conditions:

		RNA Volume
Lane 1	Before Ethanol Precipitaion	10ng
Lane 2		20ng
Lane 3		40ng
Lane 4		80ng
Lane 5	Without Ethachinmate	10ng
Lane 6		20ng
Lane 7		40ng
Lane 8		80ng
Lane 9	With Ethachinmate	10ng
Lane 10		20ng
Lane 11		40ng
Lane 12		80ng

Affect for enzymes

Data1. Restriction Enzyme Digestion and T4 DNA Ligase

λ DNA was subjected to digestion, ligation, and re-digestion using restriction enzymes (Hae III, Hind III) and T4 DNA Ligase, in the presence or absence of Ethachinmate (1 µL per 10 µL reaction mixture).

Restriction Enzyme	Hae Ⅲ		Hind Ⅲ		Lane 1: Cut Lane 2: Cut and ligase Lane 3: Cut and ligase and re-cut
Etachinmate	-	+	-	+
Result

Result

Ethachinmate did not affect the anzyme activity of restriction ligation efficiency.

Data2. Reverse Transcriptase

In the presence of Ethachinmate, [³H]dTMP was incorporated into the poly(rA)·(dT)_12-8 template using AMV Reverse Transcriptase (RTase).

Reverse Transcription Reaction

50 µL [50 mmol/l Tris-HCl(pH8.3), 6 mmol/l MgCl2, 40 mmol/l KCl, 0.5 mmol/l [³H]dTTP, 0.4 mmol/l poly(rA)・(dT)_12-8, 5 units AMV RTase]

Temperature: 42 ℃

RTase	Ethachinmate
-	10 µL
+	0 µL
+	5 µL
+	10 µL

Result

Ethachinmate did not affect the activity of Reverse Transcriptase.

Data3. Taq DNA Polymerase

In the presence of Ethachinmate, a DNA (about 600 bp) was amplified using Taq DNA Polymerase.

PCR Reaction

25 µL [TAPS-HCl, pH9.3, 50 mmol/l KCl, 2 mmol/l MgCl2, 1 mmol/l 2-Mercaptoethanol, 0.01 % gelatin, 200 µmol/l dNTPs, 0.2 M primer, 1.25 units Taq DNA Polymerase]

Lane 1 : Ethachinmate 0 µl
Lane 2 : Ethachinmate 0.2 µl
Lane 3 : Ethachinmate 0.5 µl
Lane 4 : Ethachinmate 1 µl
Lane 5 : Ethachinmate 3 µl
Lane 6 : Ethachinmate 5 µl

Result

Ethachinmate did not affect the activity of Taq DNA Polymerase.