GV-Plasmid DNA Mini Isolation kit 50T

Cat NO:GV-MN-P-50

The Kit provides a rapid plasmid purification method, based on the theory of the improved SDS alkali cracking andthe silicon substrate membrane selectively adsorbing DNA. More than 20 μg plasmid DNA can be purified from 1-5ml E.co li(OD600: 0.6~0.8). Isolated Plasmid DNA is ready for downstream molecular biology experiments such as sequencing, in vitro transcription and translation, restriction enzyme digestion,bacteria transformation.

 Kit Components, Storage, Stability

Component

Amount

Attention

RNase A

0.5 ml

-20℃

Buffer GS1

13 ml

For Plasmid

Buffer GS2

13 ml

For Plasmid

Buffer GS3

18 ml

For Plasmid

Buffer BL

25ml

For plasmid Gel Extraction/DNA Clean

Buffer W1

6 ml

Add 24 ml ethanol, For DNA washing

Buffer W2

6 ml

Add 24 ml ethanol, For DNA washing

 Elution

6 ml

For DNA Elute

Spin Columns

50个

 

1.5ml Tubes

50个

 

Handbook

1

 

 

 

 

 

 

 

RNase A: 10mg/ml, save six months at room temperature before open package, store at -20 ℃ for long-term。

Buffer GS1: Bacteria Suspension. Add RNase A and mix together, store at 4℃。

Buffer GS2: Bacteria Cracking Buffer (contain SDS/NaOH), airtight storage at room temperature.

Buffer GS3: Neutralition Buffer. Airtight stored at room temperature.

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

Buffer W1: Washing Buffer. Add ethanol (as the volume marked on bottle label) before use. Airtight stored at room temperature.

Buffer W2: Desalting Buffer. Add ethanol (as the volume marked on bottle label) before use. Airtight stored at room temperature.

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

 

Announcements

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

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

3. BufferGS2, Buffer GS3, Buffer BL contains irritant compounds, wear latex gloves and glassesavoiding contaminate skin, eyes and clothes, beware of suction to nose. If contaminate skin or eyes, please wash with plenty of water or saline immediately,seeing the doctor for advice when necessary.

 

Preparation reagent

1.     Al the RNase A should beadded into Buffer GS1 before use, mix and store at 4℃.

2.     Add ethanol (as the volume marked on bottle label) to Buffer W1 and Buffer W2 before use.

3.     If the Buffer GS2 precipitated, redissolve by warming to 37℃ and cool to room temperature before use.

4.     Warm the elution or ddH2O to 65℃ to improve elution efficiency.

 

Procedure

1.     Add 1-5ml cultured bacteria to 1.5ml micro centrifuge tube, centrifuge at 12000rpm for 1minand discard the supernatant.

    Note: Clean the supernatant as clean as possible, otherwise affect the plasmid purity.

2. Add all the RNase A into Buffer GS1(store at 4℃). Resuspend bacterial cells by 250μl Buffer GS1 until no pellet bacterial cells.

3. Add 250μl Buffer GS2 and gently invert the tube 4-6 times to mix until the solution become clear. Don’t allow this step for more than 2 minutes.

Note: Do not vortex. Mix until the solution clear is enough.

4.  Add 350μl Buffer GS3 and invert the tube 4-6 times immediately to mix. Let it stand for 2 minutes to neutralize the solution completely.

Note: The solution should become cloudy.

5.  Centrifuge for 10minutes at 12,000rpm, take the supernatant.

6.  Deal centrifugal column before the supernatant banding column: add 200ul Buffer BL to column, discard Equilibrium Buffer in collection tube.

7. Apply the supernatant from step 5 to the spin column, and stand for 2 minutes

Note: Now the centrifugal column is in collecting tube. Don’t pour sediment into centrifugal column.

8.   Centrifuge for 1minutes at 12,000rpm, discard the flow-through.

Note: Now DNA is adsorbed on silicon substrate membrane of the centrifugal column.

9.     Add 500μl Buffer W1 (add 24ml ethanol before use) , and stand for 2 minutes, centrifuge for 1minutes at 12,000rpm, discard the flow-through.

Note: Wash off impurity such as protein, salt on the membrane.

10. Add 500μl Buffer W2 (add 24ml ethanol before use) , and stand for 2 minutes, centrifuge for 1minutes at 12,000rpm, discard the flow-through.

11. Centrifuge for 2 minutes at 12,000rpm, remove residual liquid.

       Note: Remove residual ethanol for DNA dissolving at next step.

12. Transfer the column to a sterile 1.5ml micro centrifuge tube, and stand for 5-10 minutes open. Add 50100μl Elution in the middle of the silicon substrate membrane, and stand for 2 minutes at room temperature. Centrifuge for 2 minutes at 12,000rpm, plasmid DNA is in the solution at tube bottom.

 

Detection

   Plasmid extracted by the Kit can be detected by agarose gel electrophoresis and ultraviolet specrophotometer to conform 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

RNA residual

No RNase A in Buffer GS1

Add RNase A into Buffer GS1

RNase A inactivated in Buffer GS1

Store Buffer GS1containing RNase at 4℃

Extraction efficiency is low

The bacteria is cultivated too long time

Culture time should be no more than 16 h

Low plasmid copy number

Increase the amount of bacteria, and increase the dosage of GS1 GS2, GS3 in proportion according to the actual situation

Less Bacteria cells, low concentration

Increase the bacterium cells

Bacteria cells is resuspended inadequately

Add Buffer GS1 and resuspend bacterial cells adequately

No ethanol added into Buffer W1and W2

Make sure adding ethanol into Buffer W1and W2

Misuse the Washing Buffer

Use Washing Buffer 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

 

 

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