96 Well Double Stranded Sequencing Template Isolation for Sequencing with Fluorescent terminators

We earlier published a paper describing our large scale plasmid, cosmid and P1 isolation in:

H. Q. Pan, Y. P. Wang, S. L. Chissoe, A. Bodenteich, Z. Wang, K. Iyer, S. W. Clifton, J. S. Crabtree and B. A Roe. The complete nucleotide sequences of the pSacBII P1 cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and LAWRIST16. Genetic Analysis Techniques and Applications 11, 181-186 (1994).

However, recently we have investigated a procedure for large scale BAC, PAC, Cosmid, Fosmid isolation using the cleared lysate method followed by a double acetate precipitation that has been developed at the Genome Sequencing Center, Washington Univ. St. Louis, MO.

This method yields large insert target DNA that contains less than 1-2% host genomic contamination in our hands and can be used for both shotgun cloning and/or direct sequencing with universal or custom synthetic primers with the TaqFS and Big Dyes. but now are using a semi-automated double stranded sequencing template isolation as described in the protocol below.



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Latest and Greatest Protocols for double stranded template isolation and
fluorescent-labeled terminator sequencing as used in the Roe Lab - 3-21-96
Updated September 26, 1999
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	Protocol for the Manual 96 Well Double Stranded Template Isolation
                            Version dated 12-20-95
             Developed by Steven Toth in in Bruce Roe's Laboratory at the
                            University of Oklahoma.
                           Updated September 26, 1999

1. Pick colonies using a toothpick into 1.5 ml TB + salts (100 ug/ml Amp) and
   shake for 24 hours at 350 rpm in a 96 well block (Beckman #140504) with cover
   (PolyFiltronics #00-04-0006, phone 617 878-1133).
2. Harvest cells by centrifugation at 2000 rpm for 7 min.  Pour off the 
   supernatant and allow the pellets to drain inverted.  Cell pellets may
   be frozen at this point if necessary.
3. Add 200 ul TE-RNase to the cell pellets and mix by vortexing and/or
   pipetting up and down with a 12 channel pipet to resuspend. Place in 37oC
   shaker for 15 minutes.
4. Add 200 ul SDS/NaOH and shake by hand to mix.  Incubate at room temperature
   for 1 hour or until all cells dissolve by visual inspection.
5. Add 200 ul 3M KOAc, pH 4.8 (3M NaOAc, pH 4.8 works equally well) to the
   wells and vortex to mix.  Incubate  30 minutes in 37oC shaker, followed by a
   -20oC incubation for 1 hour minimum or until frozen solid (Overnight at -20
   deg C is recommended and recently [Sept.1999] shown to give a tighter pellet).
6. Centrifuge for 30 minutes at 3000 rpm in a cooled Beckman GPR centrifuge to
   pellet the precipitate.
7. Remove the top 500 ul using a 12 channel pipet to a clean 96 well block.
8. Add 1.5 ml 95% ethanol and incubate at -20oC for 1 hour to overnight.
9. Centrifuge for 30 minutes at 3000 rpm in a cooled Beckman GPR centrifuge to
   pellet the DNA.  Decant and wash three times with 500 ul 70% ethanol and
   centrifuge for an additional 5 minutes at 3000 rpm in a cooled Beckman GPR
   centrifuge.
10. Decant the supernatant, drain inverted on a paper towel.  Dry under vacuum.
11. Resuspend in 100 ul ddH2O and assay by agarose gel electrophoresis.

      TE-RNase:						SDS/NaOH:
      --------                                          --------
  1.2 ml 1 M Tris-HCl, pH 7.6			20 ml 1 N NaOH (or 0.8 g)
  480 ul 0.5 M EDTA				10 ml 10% SDS (or 1 g)
  50 ul 20 mg/ml RNase A  			ddH2O to 100 ml (make fresh)
  10 ul 100U/ul RNase T1
  ddH2O to 24 ml

  3M KOAc, pH 4.5:
  ---------------
  294.45 g  KOAc
  300 ml ddH2O to dissolve, adjust pH with 
  glacial acetic acid, bring to final volume 
  of 1 liter with double distilled water.

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		96 Well Double Stranded Template Isolation
		  	Version dated 4-29-95
         Developed by Judy Crabtree and Guozhong Zhang in Bruce Roe's
                 Laboratory at the University of Oklahoma. 

1. Pick colonies using a toothpick into 1.8 ml TB + salts (100 ug/ml Amp) and
   shake for 24 hours at 350 rpm in a 96 well block (Beckman #140504) with cover
   (PolyFiltronics #00-04-0006, phone 617 878-1133).
2. Harvest cells by centrifugation at 2000 rpm for 7 min.  Pour off supernatant
   and allow pellets to drain inverted.  Cell pellets may be frozen at this 
   point if necessary.
3. Turn on Biomek, begin the program DSISOL2 and set up the biomek as indicated
   in the configuration function on the screen. Specifically, you should put
   TE-RNase A (50:10 TE buffer containing 40 ug/ml DNase-free RNase A) in the
   first module, SDS/NaOH (1% SDS containing 0.2 M NaOH) in the second reagent
   module and 3 M KOAc, pH 4.8 in the third module.
4. Place the 96 well block containing cells onto the biomek tablet at the
   position labeled "1.0 ml Minitubes".  Place a Millipore filter plate
   (#MADVN6510) in the position labeled "96well flat bottomed microtitre plate".
5. Press ENTER to continue with the program.
6. First the biomek will add 100 ul TE-RNase A to the cell pellets and mix
   to partially resuspend. Place in 37oC shaker for 5 minutes.
7. Next, the biomek will add 100 ul SDS/NaOH to the wells of the filter plate.
8. The biomek then will mix the cell suspension again, transfer the entire
   volume to the filter plate containing SDS/NaOH, and mix again.  Set up the
   filtration apparatus with a clean 96 well block to collect the filtrate (we
   wash and reuse the block used for growth). Pause the biomek and allow to 
   incubate for 20 minutes at room temperature.
9. The biomek will add 100 ul 3M KOAc, pH 4.8 to the wells of the filter plate
   and mix at the sides of the wells.  Some choose to place the filter plate at
   -20oC for 5 minutes at this point.  Transfer the filter plate to the QiaVac
   Vacuum Manifold 96 (Qiagen Cat. No. 19504) and filter using water vacuum only
   (do not do a harsh filtration as the plates are fragile and will loose their
   seal).  This will typically take less than 20 minutes.
10. The supernatant collected in the 96 well block is the crude DNA and must be
    ethanol precipitated before use by the addition of 1 ml 95% (or 100%) 
    ethanol and incubation at -20 degC for at least 30 minutes.
11. Centrifuge for 25 minutes at 3000 rpm in a cooled Beckman GPR centrifuge.
12. Decant and wash three times with 500 ul 70% ethanol and centrifuge for an
    additional 5 minutes at 3000 rpm in a cooled Beckman GPR centrifuge.
13. Decant the supernatant, drain inverted on a paper towel.  Dry under vacuum.
14. Resuspend in 100 ul ddH2O and assay by agarose gel electrophoresis.
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