Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-15 exp2: Difference between revisions

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**Get the refence position of post-ligation reactions from internal marker template.
**Get the refence position of post-ligation reactions from internal marker template.
**Compare the effect of adding De-P-carrier or not on library construction.
**Compare the effect of adding De-P-carrier or not on library construction.
**Test the contamination of De-P-carrier


==Pocedures==
==Pocedures==
*Prepare the following samples
**E.coli internal marker (IM) (200bp): 43.6ng/uL
**1/10 dilutted E.coli internal marker (IM) (200bp): 4.36ng/uL
**DNAse digested-cleaned (D-C) tRNA 109 ng/uL
Sample name                          L1            L2          L3          L4            L5          L6
                                  200ng IM        50ng IM      10ng IM      50ng IM      10ng IM        0ng IM
                                  300ng tRNA    450ng tRNA  490ng tRNA    450ng tRNA    490ng tRNA    500ng tRNA
-----------------------------------------------------------------------------------------------------------------
E.coli IM      (43.6ng/uL)          4.59          1.146        -          1.146            -            -
1/10 E.coli IM (4.36ng/uL0            -            -        2.293                      2.293          -
D-C-tRNA      (109ng/uL)            2.75          4.128      4.49        4.128          4.49          4.587
H2O                                42.66        44.726      43.217      44.726        43.217        45.413
--------------------------------------------------------------------------------------------------------------
                                    100.00        100.00      100.00      100.00        100.00        100.00 uL
===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
*In a non-sticky 1.5-mL tube. Add 10uL 3M NaOAc, 250uL 100% EtOH, 1.5uL Glycoblue and 100uL DNA samples. Vortex 5sec.
*Incubation at 80C for 25min. Pelleting at 10,000xg 4C for 25min. Remove supernatant.
*Washing with 750uL 75%EtOH. Pelleting at 10,000xg 4C for 5min. Remove supernatant.
*Air dry the pellet. '''Dissolve the pellet in 34uL H2O'''.

Revision as of 05:56, 16 February 2010

Low-Input DNA libarary construction - Internal marker templates

Background

  • Test low-input DNA libaray conscturction using internal marker at 200ng, 50ng, 10ng at 0ng, with or without De-P-carrier.
  • Goals:
    • Test the newly synthesized adaptors and primers by myself.
    • Test Celso's protocol (with new End-repaing recipe).
    • Get the refence position of post-ligation reactions from internal marker template.
    • Compare the effect of adding De-P-carrier or not on library construction.
    • Test the contamination of De-P-carrier

Pocedures

  • Prepare the following samples
    • E.coli internal marker (IM) (200bp): 43.6ng/uL
    • 1/10 dilutted E.coli internal marker (IM) (200bp): 4.36ng/uL
    • DNAse digested-cleaned (D-C) tRNA 109 ng/uL
Sample name                           L1            L2          L3           L4             L5           L6
                                 200ng IM        50ng IM      10ng IM       50ng IM       10ng IM        0ng IM
                                 300ng tRNA     450ng tRNA   490ng tRNA    450ng tRNA    490ng tRNA    500ng tRNA
-----------------------------------------------------------------------------------------------------------------
E.coli IM      (43.6ng/uL)           4.59          1.146         -          1.146            -            -
1/10 E.coli IM (4.36ng/uL0             -             -         2.293                       2.293          -
D-C-tRNA       (109ng/uL)            2.75          4.128       4.49         4.128          4.49          4.587
H2O                                 42.66         44.726      43.217       44.726         43.217        45.413
--------------------------------------------------------------------------------------------------------------
                                   100.00        100.00      100.00       100.00         100.00        100.00 uL

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
  • In a non-sticky 1.5-mL tube. Add 10uL 3M NaOAc, 250uL 100% EtOH, 1.5uL Glycoblue and 100uL DNA samples. Vortex 5sec.
  • Incubation at 80C for 25min. Pelleting at 10,000xg 4C for 25min. Remove supernatant.
  • Washing with 750uL 75%EtOH. Pelleting at 10,000xg 4C for 5min. Remove supernatant.
  • Air dry the pellet. Dissolve the pellet in 34uL H2O.