Geneflow Q-Spin Plasmid DNA Purification Kit

  • Reproducible yield of up to 25 g of plasmid DNA per miniprep
  • Spin column format, clear instructions
  • Purified plasmid DNA is suitable for DNA sequencing, PCR, restriction enzyme digestion
  • More than 700 bases can be clearly sequenced

DHigh copy plasmid purified from 2 ml of E.coli overnight LB-culture using Q-Spin DNA Plasmid Purification Kit (5 ul/lane). DNA was eluted from spin column with 50 ul of elution buffer.

The Geneflow Q-Spin Plasmid DNA Purification kit provides a simple and efficient method for both high and low copy number plasmid DNA purification. Plasmid DNA is selectively absorbed in the spin column, while genomic DNA, proteins, salts and nucleotides are washed off.

Up to 25 g of plasmid DNA from 1-10 ml of overnight E. coli culture can be purified.

Applications

The Plasmid DNA purification kit has been optimised for efficient extraction of plasmid DNA from E. coli

The kit is suitable for high and low copy plasmids. The quality of the purified plasmid DNA is suitable for all downstream applications (DNA sequencing, restriction enzyme digestion, in vitro transcription and PCR).

Features

Spin column format enabling processing of 12 samples in just 20 minutes

Reagents supplied in kit

Spin columns, 2-ml collection tube, RNase A (5 mg/ml), Solution I (resuspension buffer), Solution II (lysis solution), Solution III (neutralisation solution), Wash solution concentrate, Elution buffer 5 mM Tris-Cl, pH 8.0

Geneflow Q-Spin Plasmid DNA Purification Kit Instructions

The Kit is designed for purification of plasmid DNA from 1-2 ml (high copy number), or up to 10 ml (low copy number) of E.coli overnight culture.

Components      
Spin Columns 3 50 250
2-ml Collection Tubes 3 50 250
Rnase A, 5 mg/ml 20ul 300ul 1.5ul
Solution I, resuspension buffer 0.5ml 12ml 60ml
Solution II, lysis solution 0.5ml 12ml 60ml
Solution III, neutralisation solution 1.0ml 24ml 2 x 60ml
Wash Solution, concentrate 1.05ml 17.5ml 5 x 17.5ml
Elution Buffer, 5 mM Tris-Cl, pH 8.0 0.2ml 5ml 25ml

Notes before use

  1. Add RNase A to Solution I. Then Solution I should be stored at +4 C.

  2. Solution II may form a precipitate. Dissolve the precipitate by warming at 37-50 C.

  3. Add 2.3 volumes of ethanol (96-100%) to 1 volume of Wash Solution Concentrate, e.g. 2.5 ml of ethanol to 1.05 ml, or 41 ml to 17.5 ml.

  4. TE buffer is not recommended for elution.

  5. Bench top micro centrifuge (7,000-12,000 rpm) can be used for all following procedures.

  6. Optimum volume of overnight culture is 2 ml, incubate with Solution I for up to 5 minutes when using bigger volumes of culture.

Protocol

  1. Pellet cells from overnight culture (1-2 ml) in 1.5 ml or 2 ml microfuge tube with centrifuging for 15 seconds. Remove supernatant completely.

  2. Add 150 ml of Solution I and vortex to resuspend the pellet.

  3. Add 150 ml of Solution II, mix by gentle inverting (3-7 times) or shaking. Do not vortex! Mixture gets clear with lysis of cells. Do not incubate for longer than 1 minute.

  4. Add 300 ml of Solution III and mix immediately. Incubate at room temperature for 1 minute.

  5. Centrifuge for 7-10 minutes.

  6. Transfer the supernatant to the column inserted into collection tube. Spin for 30 seconds.

  7. Discard the flow-through liquid. Add 500 ml of Wash Solution to the column. Spin for 15 seconds. Make sure there is no liquid in the column.

  8. Repeat wash step 7.

  9. Discard the flow-through in the collection tube. Centrifuge for 1 minute to remove residual Wash Solution.

  10. Transfer the column into 1.5 ml microfuge tube. Add 50 ml of Elution Buffer on to the center part of membrane of the column and incubate for 2 min at room temperature. Use preheated Elution Buffer (37-60 C) for higher DNA recovery. Spin for 1 min.

  11. Transfer the plasmid DNA into a clean tube and store at –20 C.

 

Troubleshooting

RNA traces in final preparation

RNase digestion was insufficient. Check culture volume against recommended volumes. Incubate with RNase for longer. Add more RNase if solution is more than 6 months old.

Low yield of plasmid DNA

a) Alkaline lysis was insufficient due to higher than recommended amount of cultured medium. Reduce culture volume or increase volumes of Solutions I, II, and III.

  b) Incubate with Elution Buffer for longer time. Add the buffer directly to the center of the column’s filter. Preheat buffer and column up to 60 C before elution step for 3-5 minutes. Centrifuge at lower speed 4,000-5,000 rpm/min

For research use only.    


 
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