Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-27

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Revision as of 01:29, 1 March 2010 by >Sam Chiang (→‎Pocedures)
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Low-Input DNA libarary construction - Internal marker templates test-2

Background

  • Repeat the low-input DNA libaray conscturction with following changes:
    • Add inactivation step following End-repaining.
    • Try two more conditions of De-P-Carriers (carriers made by SAP 60min or ACP 60min)
    • Change the order of size-selection. The size-selection will be performed prior PCR amplification.

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                          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
    • 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.

End-reparing & Phosphorylation

  • End-it Kit (EPICENTRE)
                      1rxn     8.5rxn
DNA+H2O               34.0      -
10X buf.               5.0     42.5
2.5mM dNTP             5.0     42.5
10mM  ATP              5.0     42.5
Enzyme                 1.0      8.5
------------------------------------
                      50.0    136.0   136/8.5=16 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
  • Adding De-P-Carrier into the DNA mixture during QIAquick washing.
  • Concentration of De-P-Carrier 1ug/30uL = 33.3ng/uL
    • S30: 450ng for B4 reaction: 450/33.3=13.5uL
    • S30: 490ng for B5 reaction: 490/33.3=14.7uL
    • S30 :500ng for B6 reaction: 500/33.3=15.0uL
    • S60 :500ng for B7 reaction: 500/33.3=15.0uL
    • A60 :500ng for B8 reaction: 500/33.3=15.0uL
  • Elute DNA in 28uL EB

A-tailing

  • Exo-minus klenow DNA pol. kit (Epicentre)
                      1rxn     8.5rxn
DNA+H2O               28.0      -
10X buf.               4.0     34.0
1mM dATP               6.0     51.0
Enzyme                 2.0     17.0
------------------------------------
                      40.0    102.0   102/8.5=12 
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
  • QIAquick purification. Eluted in 20uL EB.