Sam:LabNotes/Microbiome-new/2010-3-2

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Revision as of 06:24, 3 March 2010 by >Sam Chiang (→‎Procedures)
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Single-bacterium DNA library construction - test 1

Background

  • Both of following protocols were working for low-input DNA library construction:
    • Size selection prior PCR amplification (with/without De-P-Carrier)
    • Size selection after PCR amplification (with/without De-P-Carrier)
  • I decided to use the method with size selection prior PCR amplification, but without adding De-P-Carriers.
  • This experiment also includes the tests of different amount of positive control using sheared DNA.

Procedures

  • Prepare the following samples:
    • DNAse digested-cleaned (D-C) tRNA 83ng/uL
    • Ecoli DNA (EDNA)
    • Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)
Sample name                        C1            C2           C3            C4            C5             C6     
                                 200ng EDNA      50ng EDNA    10ng EDNA     50ng P11-1    50ng P12-1     50ng IM 
                                 300ng tRNA     450ng tRNA   490ng tRNA    450ng tRNA    450ng tRNA     450ng tRNA   
--------------------------------------------------------------------------------------------------------------------
Sheared DNA                         50.00         50.00       50.00        50.00          50.00           -
E.coli IM      (43.6ng/uL)             -            -           -            -              -            1.15
D-C-tRNA       (83ng/uL)             3.60          5.42        5.90         5.42           5.42          5.42

H2O                                 46.40         44.58       44.10        44.58          44.58         93.43
---------------------------------------------------------------------------------------------------------------------
                                   100.00        100.00      100.00       100.00         100.00        100.00 uL
  • Purify the DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.