(Schisto Genome Network Homepage)

BAC Sequencing Protocol

This protocol details David Williams' technique for generating BAC end sequences to use as the source of probes for the S. mansoni Physical Mapping Initiative. It was compiled by David Williams and Brad Foulk (16/12/99)

Isolation of BAC DNA using QIAGEN Plasmid Midi Kit

  1. Pick a single BAC colony and inoculate a 5 ml overnight starter culture in LB with 12.5 g/ml chloramphenicol
  2. Add 0.5 ml of the starter culture to 100 ml of LB and chloramphenicol and grow for 14 hours at 37oC with vigorous shaking.
  3. Divide the culture between two 50-ml tubes and harvest them by centrifugation at 4500 x g for 20 min.
  4. Resuspend each bacterial pellet in 10 ml (total) Buffer P1. (Ensure that Rnase A has been (100mg/ml) has been added to Buffer P1.)
  5. Add 10 ml Buffer P2 to each tube. Mix by inverting 4-6 times and incubating at room temperature for 5 min. (Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, warm buffer to 37oC to dissolve the SDS.)
  6. Add 10 ml chilled buffer P3 to each tube. Immediately mix by gently inverting 4-6 times, and incubate on ice for 15 min.
  7. Centrifuge at > 20,000 x g for 30 min at 4oC. Remove supernatant containing DNA promptly.
  8. Centrifuge at > 20,000 x g for 15 min at 4oC. Remove supernatant promptly.
  9. Equilibrate a Qiagen tip 100 by applying 4 ml buffer QBT and allow the column to empty by gravity flow.
  10. Pool the two supernatants from step 8. Apply the sample to the QIAGEN tip and allow it to enter the resin by gravity flow
  11. Wash the QIAGEN tip with 2 x 10 ml buffer QC.
  12. Elute the DNA with 5 x 1 ml aliquots of buffer QF, pre-warmed to 65oC. Pre-warming the elution buffer may help to increase yields. Eluting in 5 aliquots helps keep the eluting buffer warm.
  13. Shear the DNA by passing through a 23 gauge 1 inch needle 7 times.
  14. Precipitate the DNA by adding 3.5 ml of room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at >15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.
  15. Wash the pellet in 70% ethanol and centrifuge at > 15,000 x g for 10 min. Decant the supernatant.
  16. Air dry the pellet for 5-10 min and dissolve in 10mM Tris Cl pH 8.5.
  17. Quantitate the BAC DNA by running an aliquot on a 0.8% agarose gel and comparing it to DNA standards.

Notes:

  1. Protocol largely from Qiagen Plasmid Protocol: Isolation of BAC DNA (TS-QP1 05/98).
  2. Recipes for buffers and solutions are described in Qiagen tip 100 manual.
  3. Steps 7 & 8 may be eliminated by the use of the QIAfilter Plasmid Kits.
  4. Additional information avaiable online from Qiagen

BigDye Terminator Sequencing protocol for BACs.

Protocol derived from PE Biosystems User Bulletin: ABI Prism® 377 DNA Sequencer and ABI Prism® 310 Genetic Analyzer; Sequencing Large DNA Templates. September 29, 1998. Also available online from ABI

  1. Heat DNA at 55-60oC for 30 minutes before adding to the reaction mix.
  2. Set up the sequencing reaction as below:
    Terminator ready reaction mix 16.0 µl
    Primer 6.0 ­13.0 pmol
    BAC DNA 400-800 ng
    deionized water q.s. to 40 µl
  3. Mix contents and cycle with the following conditions:
    95oC for 5 min.
    35 to 50 cycles of 95oC - 30 sec
    50-55oC - 20 sec
    60oC 4 min.
  4. Clean up cycling reaction using a CENTRI-SEP column. Protocol available online from Princetown Separations
  5. (for ABI 310) resuspend in 15 ml template suppression reagentand run on sequencer.

(Schisto Genome Network Homepage)

updated 13/03/00