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

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Revision as of 16:48, 25 September 2010 by >Sam Chiang (→‎Sonification)
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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

Operation manual

Hook up H20-tubes of cooling system. Release the white clipper.
Fill up H2O(tap H2O is acceptable) in the cup-horn to match the yellow tags
Turn on the switch of the cooling system.
Cooling system setting: Pump speed: "50", Desired temp "4C".
Turn on the switch of main controller.
Program setting:
         o Press "No" for not using micro-tip.
         o Press "program" to set up new conditions.
         o Tested condition: Pulse on(30sec), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min). 
Press "start" to start the shearing.
Press "pause" to pause the shearing and aliqate sheared samples for validation. 

After using, use the opposite order of operation procedures to turn off the machine.
         o Press "Stop" to stop shearing.
         o Turn off the main controller.
         o Turn down pump speed to "0" and turn off the switch.
         o For the cooling system, disconnect the H2O-tubes. Drain the H2O into the beaker.
         o Wipe out the spilt H2O.