Ethanol precipitation of small DNA fragments

From OpenWetWare

Revision as of 02:38, 1 October 2009 by Vaishnavi Ananth (Talk | contribs)
Jump to: navigation, search
back to protocols

This protocol is for a simple ethanol precipitation of small fragments. This protocol was used to (partially) purify a DNA fragment containing a ribosome binding site (~40 bp) during 3A assembly. The fragment was generated via restriction digest and it was used in a ligation reaction. Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful. All DNA fragments from your digest will still be present in your pellet. These residual DNA fragments do not matter for 3A assembly which selects against incorrect ligation products.

Contents

Materials

  • Absolute Ethanol (100% = 200 proof)
  • 95% ethanol
  • Tabletop centrifuge
  • -80°C freezer

Procedure

  1. Add 2 volumes ice cold absolute ethanol to sample.
    Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.
  2. Incubate 1 hr at -80°C.
    The long incubation time is critical for small fragments.
  3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
  4. Remove supernatant.
  5. Wash with 750-1000 μL room-temperature 95% ethanol.
    Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.
  6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
  7. Let air dry on benchtop.
    I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
  8. Resuspend in an appropriate volume of H2O.
    Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.

BioStream version

Following is the Ethanol precipitation of small DNA fragments protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Ethanol precipitation of small DNA fragments protocol

Source Code

Ethanol precipitation of small DNA fragments protocol - source code

Personal tools