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Latest revision as of 06:58, 19 October 2016

Gel Extraction · Benchling

Gel Extraction

Introduction

The extraction kit is designed to recover or concentrate DNA fragments (100bp ­ 10kb) from agarose gels. Chaotropic salt is used to dissolve agarose gel and denature enzymes. DNA fragments in thechaotropic salt are bound by the glass fiber matrix of the spin column. Contaminants are removed with awash buffer (containing ethanol) and the purified DNA fragments are eluted by a low­salt elution buffer orTE. Salts, enzymes, and unincorporated nucleotides can be effectively removed from the reaction mixturewithout phenol extraction of alcohol precipitation. Typically, recoveries are up to 90% for gel extraction,and up to 95% for the PCR Cleanup Reaction. The entire procedure can be completed in 20 minutes and theeluted DNA is ready for use in PCR, fluorescent or radioactive sequencing, restriction enzyme digestion,DNA labelling and ligation. For users who require a higher recovery of small base pair DNA fragments(50­200 bp) or large base pair DNA fragments (>8 kb), another extraction kit must be used. SAFETY PRECAUTIONS SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Materials

  • Reagents
    • DF buffer
    • W1 buffer
    • Wash buffer
    • Elution buffer
  • Equipment
    • UV illuminator
    • Razor
    • 1.5 mL microcentrifuge tube
    • Vortex
    • DF column
    • 2 mL collection tube

Procedure

  • Gel Dissociation
  1. Position the agarose gel on the UV illuminator, and raise the UV shield. Have someone turn off thelab incandescent lighting, then turn on the UV light source, standing with your body and eyes behindthe UV shield to avoid damage.
  1. Use one of the razors supplied at the EtBr bench to excise the agarose gel slice containing the relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice.
  1. Weigh the gel slice, and transfer up to 300 mg to a 1.5 mL microcentrifuge tube. Add 500 μL of DFbuffer to the sample and mix by vortex.
  1. Incubate at 55 to 60 degrees Celsius for 10 to 15 minutes or until the gel slice has been completelydissolved. During incubation, invert the tube every 2 to 3 minutes.
  1. Cool the dissolved sample mixture to room temperature.
  • DNA Binding
  1. Place the DF Column in a 2 mL collection tube.
  1. Transfer 800 μL of the sample mixture from the previous step to the DF Column.
  1. Centrifuge at 14­16,000 x g for 30 seconds.
  1. Discard the flow­through and place the DF Column back in the 2 mL collection tube. If the sample mixture is more than 800 μL, repeat the DNA binding step.
  • Wash
  1. Add 400 μL of W1 buffer into the DF Column.
  1. Centrifuge at 14­16,000 x g for 30 seconds and discard the flow­through.
  1. Place the DF Column back in the 2 mL collection tube.
  1. Add 600 μL of wash buffer (ethanol added) into the DF Column and let stand for one (1) minute.
  1. Centrifuge at 14­16,000 x g for 30 seconds and then discard the flow­through.
  1. Place the DF Column back in the 2 mL collection tube.
  1. Centrifuge at 14­16,000 x g for 3 minutes to dry the column matrix.
  • DNA Elution
  1. Transfer the dried DF Column to a new 1.5 mL microcentrifuge tube.
  1. Add 20­50 μL of elution buffer or TE into the centre of the column matrix.
  1. Let stand for two (2) minutes or until the elution buffer/TE is absorbed by the matrix.
  1. Centrifuge for two (2) minutes at 14­16,000 x g to elute the purified DNA.
  • PCR Clean­ Up Reaction Protocol
  • Sample Prep
  1. Transfer up to 100 μL of a reaction product to a 1.5 mL microcentrifuge tube.
  1. Add 5 volumes of DF Buffer to 1 volume of the sample and mix by vortex.
  • DNA Binding
  1. Place a DF Column in a 2 mL microcentrifuge tube.
  1. Transfer the sample mixture to the DF Column and centrifuge at 14­16,000 x g for 30 seconds.
  1. Discard the flow­through and place the DF Column back in the 2 mL collection tube.
  • Wash
  1. Add 600 μL of wash buffer (ethanol added) into the centre of the DF Column and let stand for one(1) minute.
  1. Centrifuge at 14­16,000 x g for 30 seconds.
  1. Discard the flow­through and place the DF Column back in the 2 mL collection tube.
  1. Centrifuge again for three (3) minutes at 14­16,000 x g to dry the column matrix.
  • DNA Elution
  1. Transfer the dried DF Column to a new 1.5 mL microcentrifuge tube.
  1. Add 20­50 μL of elution buffer or TE to the centre of the column matrix.
  1. Let stand for two (2) minutes or until the elution buffer/TE is absorbed by the matrix.
  1. Centrifuge for two (2) minutes at 14­16,000 x g to elute the purified DNA.
  • Notes
  • Troubleshooting
  • Low Yield: a. The problem may be a poorly­dissolved gel slice ­ the gel slice may be too big; if using more than 300 mg of gel slice, separate it into multiple tubes. Additionally, one may raise the incubation temperature to 60 degrees Celsius and extend the incubation time. b. The problem may be an incorrect elution step ­ Ensure that the Elution Buffer is added to the centre of theDF column matrix and is completely absorbed. c. The problem may be an incomplete elution step ­ If plasmid DNA is larger than 10kb, use pre­heated elution Buffer (65-­70 degrees Celsius) in the elution step. Eluted DNA does not perform well in downstream applications (i.e. PCR, digestion, ligation, sequencing): d. The problem may be residual ethanol contamination ­ Following the wash step, dry the DF column with additional centrifugation at 14­16,000 x g for 5 minutes, or incubate at 60 degrees Celsius for 5 minutes. The problem may be that DNA was denatured (a smaller band appeared on gel analysis) ­ Incubate the eluted DNA at 95 degrees Celsius for 2 minutes, and then cool down slowly to re­anneal the denatured DNA.
  • Safety
  • Please handle the razor used for excision of the agarose gel slice with care; in addition, ensure that the UV shield is raised when using the illuminator to discern where the gel bands are. For PCR Cleanup Reactions, add 70% EtOH to the liquid waste generated from the reaction, and pour down the drain, with the water running for at least one minute after the pour. This allows for dilution of the chemicals poured down the drain. For gel extraction waste, add all liquid waste to the green EtBr waste bin located beneath the EtBr bench.
  • Medstore Ordering Info
  • Gel/PCR Fragment Extraction Kit (100 preps, FroggaBio), DF100 - $99.00
  • References
  • The hard copy of the protocol is kept on the literature desk, closest to the South wall.
  • Retrieved from "http://local.biochemistry.utoronto.ca/igem/index.php?title=Gel_Extraction&oldid=908"
  • Changelog
  • 1. Created 10/5/2016



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