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> MANUAL SEQUENCING METHODS (ANCIENT HISTORY)

> NEB Sequencing (Vent Polymerase)-- Preparing Samples to be Sequeneced

   

> NEB Extension/Termination 35S-dATP Sequencing

   

> Sequencing Gels

    > Sequencing Reaction: (Sequenase)

NEB Sequencing (Vent Polymerase)-- Preparing Samples to be Sequeneced

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  1. Most DNA that we sequence is in plasmid form or is PCR product.  This DNA is double stranded and therefore the DNA must be denatured prior to sequencing.
  2. To denature the DNA add to each tube:

    4M NaOH 1.0uL
    4mM EDTA 1.0uL
    dH2O 15.5uL
    DNA 2.5uL
    Total 20.0uL
  3. Incubate at room temp. For 5 min.
  4. Ethanol precipitate the solution to pellet the DNA.  This step gets rid of the NaOH.

    Steps:

    Add 4uL of 5 M NH4OAc
    Add 2X the volume of EtOH(100%) = 48uL EtOH 
    Spin in microcentrifuge for 15'
    Keep the pellet (hard to see), discard supernatant.
    Allow pellet to dry under vacuum (to get rid of any traces of the ammonium acetate.
  5. Add 6.5uL of dH2O to resuspend pelleted DNA.
  6. Heat at 95 C for 5' (to denature local secondary structures that form in the DNA since the NaOH has been removed.
  7. After 5' the tubes are placed in an ice bath to "quick-cool" them. (This ensures denatured DNA will not reanneal)
  8. Anneal DNA to primer.
    DNA 6.5uL
    10X vent buffer 1.5uL
    primer (T7 5pmol/uL) 1.0uL
    Total 9.0uL
  9. Incubate at 42 C for 20'.
  10. Extension reaction--add the following to the above;
    Ext. Mix (25uM dTTp, dCTP, dGTP) 2.0uL
    35S dATP 2.0uL
    Vent polymerase 1.0uL
    DNA + primer solution 9.0uL
    Total 14uL
  11. Incubate at 42 C for 5'.
  12. Prepare four tubes labeled G,A,T and C for each DNA being sequenced. Start adding 3uL of each dNTP solution into its respective G,A,T or C tube.
  13. Add 2.8uL of extension reaction (from the total of 14uL that you prepared) to each to the  G,A,T and C tubes for that DNA. Mix by flicking with finger or vortex gently, incubate at 72 C for 10'. (The extension reaction proceeds very quickly ~ 30bp/sec)
  14. This solution is spun in a microcentrifuge to bring down any condensate on the tube and 4uL of STOP/LOADING solution is added to each tube. The tubes can then be frozen and kept at -20o C for 1 week.
  15. The samples should be heated before loading at (72 C for 2 min or 7 min if previously frozen), so that they are the same temperatue as the gel.
  16. Insert the combs and begin running the gel in order to warm it up.  Before loading samples it is necessary to clear the urea out of the wells.
  17. Leave the tubes in the heat block and add 2.0uL from each tube (G,A,T and C) to the wells of the sequencing gel.