Sam:LabNotes/Microbiome-new/2010-9-24

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Cloning Sanger sequencing test on E.coli gDNA MDA amplicons

Background

  • To optimize the Sanger sequencing protocl for MDA amplicon validation
  • This protocol will also include the S1 nucelase debranching, DNA Pol.I reparing procedures, and compare the difference between with and without these treatments.

Plan

  • Prepare 2ug of E.coli gDNA MDA amplicon for each reaction in 100uL volume
  • S1 digestion(20units) for 0min(no S1 enzyme), 30min, 60min
  • Aliquote 80uL of digested DNA for sonification into 1~2 kB (Keep another 20uL for comparison)
  • Agarose gel size select the 1~2kB region. Recover DNA using Zymo kits or Montage filter.

Procedures

S1 nuclease digestion (debranching)

                     1rxn      6.5rxn
 DNA(666.ng/uL)       3.0      19.5
 S1(10U/uL, USB)      2.0       -
 2X S1 rxn buffer    50.0     325.0
 H2O                 45.0     292.5
 ----------------------------------
                    100.0     637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
  • Incubate at PCR thermocycler for 30min(2rxn) and 60min(2rxn).
  • When incubation is completed, inactivate the reaction by adding 1uL 0.5M EDTA, vortex mixing, and incubating at 85C for 3min.
  • Store the S1-treated DNA in -20C.

Sonification

Sonification program:

Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min).
  • Store the sheared DNA in -20C

Agarose gel size selection (with Zymo gel extraction kit)(09-26-'10)

  • The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel.
  • Incubate at 55C in hotplate for 10min (with 500 rpm agitation). Briefly vortexing to mix the dissolved gel.
  • Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through.
  • Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through.
  • Repeat the washing step again.
  • Add 15uL pre-warmed clean water on the filter. Incubate at RT for 1min. Transfet the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA.