Precipitation Protocol After Ligation

2015-01-13

azim58 - Precipitation Protocol After Ligation

Precipitation Protocol After Ligation
Note that whenever I have precipitated DNA, I cannot obtain a good
reading on the nanodrop. I am not sure if this is because of the leftover
glycogen or I may not have let the ethanol dry away completely.

* Add water, acetic acid, a carrier molecule, and ethanol
  o Start thawing glycogen and ultrapure H20 from kit
    - glycogen last seen on Felicia's -20 C shelf) or Andrey's
      strategic reserve box -20 C

o Transfer volume to larger tube (1.5 mL)
  o Increase volume to 100 uL with water
  o Add 1/10 volume (10 uL) 3 M NaAc pH 5.2 (last seen Felicia's
    shelf at room temp)
    - The sodium acetate acts as a salt to help precipitate the DNA
      or RNA

o Add a carrier molecule
    - Either
      @ Add 2 uL 20 ug/uL glycogen
      @ Note that if you need to scale up the reaction for a
        larger volume, still just use 2-3 uL of glycogen even though
        everything else will be scaled up

- Or
      @ Add tRNA to a working concentration of 10-20 ug/mL
        # Some people feel that tRNA is a higher quality
          carrier than glycogen. It has the advantage of not interfering
          with interactions between DNA and proteins like glycogen does,
          but has the disadvantage of interfering with any reactions
          catalyzed by polynucleotide kinase or terminal transferase.
        # tRNA last seen 10-3-11 KW -20 C box

o Add 3 volumes (300 uL) 95-100% ethanol
  o Vortex to mix everything up

* Cold Precipitation
  o Place tube at -80 C for 30 min- couple of hours or -20 C
    overnight/over weekend

* Centrifuge 14,000 rpm 4 C 15-20 min
  o 20817 rcf in Eppendorf centrifuge at room temp next to Debbie's
    bench. The current centrifuge in cold room may not spin fast enough
    so it may be best to use the one at room temp

* Decant supernatent
* Briefly spin down again in a small centrifuge. Decant supernatant.
* Wash pellet
  o Wash with 70% ethanol (with about 100 uL).
    - If the pellet is small (like a pellet from about a 20 uL
      ligation reaction), you may want to transfer the liquid and pellet
      to a smaller PCR tube at this point since the pellet will only be
      resuspended in 1 uL.
      @ Make sure pellet gets transferred. May need to pipette up
        and down to loosen pellet from surface. May want to use 1 mL
        pipette tip.
        # Note that the 5 mL pipette tips cannot fit into 1.5
          mL tubes

- If the pellet is large (like the pellet from about a >50 uL
      ligation reaction), you may want to leave the pellet in the same
      tube it is in since it is harder to transfer the pellet without
      losing some of it, and you may want to finally resuspend in a
      larger volume (like 2 uL).

o Gently flick tube to wash pellet
  o Centrifuge at 14,000 rpm @ 5 min
    - PCR tube can be centrifuged by placing in a 0.6 mL tube
      placed in a 1.5 mL tube

o Carefully remove supernatent
  o Briefly spin down again in a small centrifuge. Decant supernatant.

* Air dry for about 10 min (pellet goes clear)
  o May want to air dry in hood to avoid any contamination

* Resuspend in 1 uL water (maybe 2 uL for a larger pellet from a >50 uL
  reaction)
  o May want to use ultra pure H20 from a 1.5 mL tube.
  o Note that if the sample was resuspended in a larger volume than 2
    uL that the sample concentration can be determined with a nanodrop if
    desired. (see determining concentration of dna for further notes)

* May want to transform bacteria with the ligation product if that is
  the next step for the procedure.
  o Electroporation or Chemical Transformation Protocol

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Questions about Protocol
Is it possible to do an in-fusion reaction after an ethanol precipitation?
Yes it is. I have evidence of an in-fusion working after an ethanol
precipitation here: 1-23-13 Sequencing results

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