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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Extraction and Purification

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Editor:  Branch: Protocol-Wiki Type: Protocol Intention: For Publication Read/Write Permissions: Open Access

Authors : 27.09.2009 -

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A low cost method for the purification of DNA fragments from agarose gel.  Such fragments can then be used for cloning, PCR or other molecular biological purposes.

If you use this protocol and have either comments or improvements, please Edit either the Article or the Discussion. If major improvements can be made, we will publish the article as an update. Authorship is of course assumed for all significant ideas or amendments.

Contents 1 List of Materials and Solutions 2 Step by Step Method 2.1 Preparation of silica solution 2.2 DNA purification from agarose gel 3 Technical Notes 4 References

[edit] List of Materials and Solutions

Silica solution (preparation instructions described below):

• Finely ground amorphorous silica particles (Sigma S-5631)
  
• 0.1M HCl
  
• LMP Buffer Solution: 6M Guanidine HCl, 0.1M NaAcetate, 1mM EDTA pH5
  

DNA Purification:

• LMP (Low Melting Point) Buffer Solution: 6M Guanidine HCl, 0.1M NaAcetate, 1mM EDTA pH5
  
• HMP (High Melting Point) Buffer Solution: 6M GuSCN, 0.1M NaAc, 2.5mM EDTA pH5
  
• Silica solution (as described)
  
• Silica Wash Solution: 55%EtOH, 70mMNaCl, 10mMtris, 2.5mM EDTA
  (425ml normal saline, 10ml 1M tris pH7.4, 5ml 0.5M EDTA pH8, 550ml 96% ethanol)
• TE
  

[edit] Step by Step Method

The first time using this method requires the preparation of the silica solution.  This can take a few hours, but once prepared, it can be kept out of the fridge almost indefinately for use in subsequent purifications.

[edit] Preparation of silica solution

1. Weigh out 5g silica (Sigma S-5631)
2. Mix with 40ml 0.1M HCl
3. Allow to settle for 2 hr
   
4. Suck off S/N and any fine particles at surface
   
5. Repeat from 2.
   
6. Resuspend in final vol 50ml with 6M GuanidineHCl, 0.1M NaAcetate pH5, 1mM EDTA
   
7. Store @ r.t. forever.

[edit] DNA purification from agarose gel

1. Under UV light, cut DNA from gel trying to remove as much surrounding gel as possible
2. Add 200µl (or 2.5 x gel volume) LMP or HMP Buffer Solution (depending on LMP or HMP gel used)
   
3. Heat 65⁰C for 10' (until melted, longer for HMP)
   
4. Add 5-10µl silica (if small fragment e.g. <300bp, keep warm) and vortex 30”
   
5. Spin briefly at high speed and remove solution without disturbing silica pellet
   
6. Wash 2X with Silica Wash Solution
   
7. Spin pellet and remove any remaining wash buffer
   
8. Resuspend in 10µl TE.
   
9. Spin and keep S/N.

[edit] Technical Notes

While this method can be used for low and high melting point gels, there are several advantages for using low melting point gels to purify fragments where cost allows.

[edit] References

This method is an improved method described previously: Boyle JS, Lew AM. (1995) An inexpensive alternative to glassmilk for DNA purification. Trends Genet. 11:8