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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 Extension/Termination 35S-dATP Sequencing

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Denature dsDNA only (use 2.5ug DNA)
4M NaOH 1.0uL
4mM EDTA 1.0uL
dH2O 15.5uL
DNA 2.5uL 20.0uL
  1. Incubate at room T for  5 minutes
  2. Ethanol precipitate.
  3. Resuspend in 6.5uL dH2O
  4. Anneal DNA to primer
    Denatured DNA 6.5uL
    -primer 10ug/uL stock
    10X Vent buffer 1.5uL
    -dsDNA ~2ug(per reaction)
    Primer 1.0ul
    -ssDNA~1ug(per reaction)
    -ssDNA: 65C 5'
  5. cool to 42C for ~ 20 minutes
  6. Mix.
  7. Incubate at 42C for ~ 20 minutes
  8. Prepare G A T C tubes; one set for each clone to be sequenced. Add 3uL of each V-1 mix to respectively labeled tubes on ice. 
  9.  Extension reaction:
    Add to annealing reaction:
    2.0uL extension mix v (1Xext)
    2.0uL 35S-dATP
    1.0uL vent (exo-) DNA polymerase (2000u/mL)
  10. Mix.
  11. Incubate 42C for 5 minutes.
  12. Add 3.2uL of extension reaction to each GATC tube, mix. Incubate at 72C for 10 minutes.
  13. Spin and quickly add 4 uL of STOP/LOADING solution.
  14. Samples may be frozen at -20C overnight before running on the sequencing gel. Heat samples 2 minutes at 72C just prior to loading. (In other words, the samples as well as the sequencing gel should be hot in order to get good seperation.) Load 2.5 uL of each GATC sample in that order. If you avoid the outermost lanes, you can load 13 loads onto one sequencing gel.

    NEB SEQUENCING (VENT POLYMERASE)--ALTERNATE METHOD PREPARING SAMPLES TO BE SEQUENCED

    1. Vectors are double stranded and firstly the DNA must be denatured. 
      For each tube:
      4 M NaOH 1.0 uL
      4 mM EDTA 1.0 uL
      dH2O 15.5 uL
      DNA 2.5 uL
      Total = 20 uL
    2. Incubate at room T for 5 minutes.
    3. Ethanol precipitate solution to pellet DNA--gets rid of NaOH in solution.
      4uL of 5 M NH4OAc
      2X the volume of EtOH(100%)=48uL EtOH (b/c 20uL solution +4 uL salt (NH4OAc) +24uL)
      spin for 15 minutes
      Keep the pellet (hard to see), discard supernatant.
    4. Each tube containing pellet was filled with 6.5uL of dH2O to resuspend pellet.
    5. Each tube is heated at 95C for 5' (to denature DNA since NaOH has been removed.
    6. After 5' the tubes are placed in an ice bath to "quick-cool" them. (This ensures denatured DNA will not reanneal)
    7. Anneal DNA to primer.
      DNA 6.5uL
      10X vent buffer 1.5uL
      primer(T7 5pmol/uL) 1.0uL
      Total 9.0uL
    8. Incubate at 42C for 20 minutes.
    9. Extension reaction--add the following to the above;
      Ext. Mix 2.0uL
      35S dATP 2.0uL
      Vent polymerase 1.0uL
      DNA + primer solution 9.0ul
      Total 14uL
    10. Incubate at 42C for 5 minutes.
    11. GATC tubes were prepared for each clone
    12. Add 3uL of each dNTP solution into its respective GATC tube.
    13. Add 2.8uL of extension reaction (14uL) for each clone to its respective GATCset of tubes. The tubes were then mixed by flicking with finger or vortex, and were then incubated at 72 C for 10'. (The reaction proceeds very quicly ~ 30bp/sec)
    14. The solution were spun and 4uL of STOP/LOADING solution was added to each solution. The tubes can then be frozen.
    15. . The samples were heated before loading at 72C for 2 minutes (or 7 minutes if previously frozen) , so that they are the same temperature as the gel.
    16. The combs were inserted into the warming gels.
    17. 2.0uL of each clones GATC tubes (while still in the heat block) were quickly added to the wells.