Sam:LabNotes/Microbiome-new/2010-3-2: Difference between revisions

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Sample name'''                          B1            B2          B3          B4            B5            B6          B7            B8'''
                                  200ng IM        50ng IM      10ng IM      50ng IM      10ng IM        0ng IM        0ng IM        0ng IM
                                  300ng tRNA    450ng tRNA  490ng tRNA    450ng tRNA    490ng tRNA    500ng tRNA    500ng tRNA    500ng tRNA
---------------------------------------------------------------------------------------------------------------------------------------------
E.coli IM      (43.6ng/uL)          4.59          1.15        -          1.15            -            -            -              -
1/10 E.coli IM (4.36ng/uL0            -            -        2.29          -            2.29          -            -              -
D-C-tRNA      (78ng/uL)            3.85          5.77        6.28        5.77          6.28        6.41        6.41          6.41
H2O                                91.56        93.08      91.43        93.08          91.43        93.59        93.59          93.59
---------------------------------------------------------------------------------------------------------------------------------------------
                                    100.00        100.00      100.00      100.00        100.00        100.00      100.00        100.00 uL
   
   
'''NOTE: reactions B1, B2 and B3 will be added with De-P-carrier. Reactions B4 - B8 won't.'''
*Purify the DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.
*Purify the DAN mixture with EtOH precipitation
**mimic the real precedures to remove the enzyme and buffers from WGA reactions
*Each sample (PCR product) has 100uL volume. Air dry the pellet. Dissolve the pellet in 34uL H2O.

Revision as of 06:04, 3 March 2010

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
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.