GV-High-Efficiency Agarose Gels DNA Purification Kit

Cat No:GV-GX-50

This kit can recover DNA fragments from various concentrations agarose gel excluding impurities such as salt, protein, RNA. For longer than 500bp fragments, the recovery is above 90%.For 200 bp-500bpfragments, the recovery is above 70%.For 80 bp-200bpfragments, the recovery is about 60%. The Kit can recoversingle, double chain and ring plasmid DNA fragments.

The kit apply an unique balance liquid treated adsorption membrane, a reagent that can activate the silicon substrate membrane will improve adsorption ability and enhance uniform and stability of the column. The membrane can specially absorb the DNA.It can also eliminate the bad environmental factors such as heat, moisture to adsorption column.

 Kit Components, Storage, Stability

Component

Amount

Attention

Buffer GA

60 ml

 

Buffer BL

12ml

 

Wash buffer

13 ml

Add 52ml ethanol before use

Elution

4 ml

 

Spin Columns

50

Max adsorption up to 20μg each

1.5ml centrifuge tube

50

 

Handbook

1

 

 

 

 

 

Buffer GA : Gel Lysis Buffer, airtight storage at room temperature. Warm up to 65℃ and cool down to room temperature before use if appear precipitation.

Buffer BL : Equilibrium Buffer. Equilibrium solution can activate silicon substrate membrane and improve adsorption ability, uniform and stability of the column. It makes the membrane single adsorb the DNA. It can also eliminate the bad environmental factors such as heat, moisture to adsorption column

Wash Buffer : Desalting Buffer. Add ethanol as the volume marked on bottle label before use, mix completely. Airtight stored at room temperature.

Elution:10mM Tris-HCl, pH8.0. Airtight stored at room temperature.

 Announcements

1. Warm theElution or ddH2O to 65 ℃to improve recovery efficiency.

2. DNA store at 10 mM Tris-HCl, pH8.0.

3. Use the column treated byequilibrium buffer the very day, don’t storage for long time.

4. In step 1, cut gel into small pieces can greatly shorten melting time(linear DNA are easy hydrolysis exposure  to high temperature condition long time), so as to improve the recovery. Don'texpose the gel containing DNA under UV to reduce damage of UV to DNA long time.

 Preparation reagent

Add 52ml ethanol into Wash Buffer before use.

 Procedure

1. Cut off agarose gel pieces containing DNA fragments as small as possible. Add Buffer GA according to the weight ratio1:3 (add 300μlBuffer GA for 100 mg agarose gel)

Note: For DNA fragments less than 300bp,Add Buffer GA according to the weight ratio1:5.

2. Warm to 55 ~ 60 ℃ for 10 min to dissolve the gel completely, oscillate 2-3 times during water bath.

3. Deal Spin column before the supernatant binding column: add 200ul Buffer BL to column, discard Equilibrium Buffer in collection tube.

4. Add the solution from Step 2toSpin column, let it stand for 2 minute, centrifuge for 30s at 12,000rpm. Add twice if the volume is too large to be added once, discard the flow-through.

5.  Add 500μl Wash buffer (add 52ml ethanol before use) to the Spin Columns, let it stand for 2 minutes, centrifuge for 30s at 12,000rpm, discard the flow-through.

6. Repeat step 5.

7. Centrifuge for 1 minute at 12,000rpm to remove residual ethanol.

8. Transfer the column to a new sterile 1.5ml micro centrifuge tube, place the centrifugal tube open for 5 ~ 10 mins to volatilize ethanol.

9. Add 2060μl Elution (pre-warm to 65℃), let it stand for 2 minutes.

10. Centrifuge for 2 minutes at 12,000rpm, purified DNA is in the solution at tube bottom, store at -20℃.

 Detection

DNA purified by the Kit can be detected by agarose gel electrophoresis and ultraviolet specrophotometer to test the concentration and purity.

DNA hasabsorption peak at 260nm. Make OD260 valuebetween 0.1 and 1.0 during testing. OD260=1 is equivalent to about 50 μg/ml double-stranded DNA, 40 μ g/ml single DNA.

Nucleic acid concentration(μg/ml)=OD260×50×dilution ratio.

OD260/OD280 value should be 1.7~2.0.

 Troubleshooting

Problem

Cause

Solution

Low recovery or no band

Purified DNA is not suitable for experiment

Melt the gel incompletely

Extend melting time and oscillate sometimes to melt completely

agarose gel is too much

Less than 300mg gel in each tube

Have not add ethanol into Wash buffer

Add 52ml ethanol into Wash Buffer

Misuse the Elution buffer

Use Elution offered by the Kit

Elution don't fully

Make sure enough elution time and pre-warm Elution to 65℃

High pH of electrophoresis buffer

Use fresh buffer electrophoresis to test sample

Little sample, low concentration

Increase the volume of sample

Problem

Ethanol residue

Extendairing time appropriately when the room temperaturelow.Put it in 37 ℃incubator for drying.

Salt residue

Make sure washing dosage and washing times is enough.

 

 

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