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

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                       1rxn    6.5rxn
                       1rxn    6.5rxn
  DNA                   34.0      -
  DNA+H2O              34.0      -
  10X buf.              5.0    32.5
  10X buf.              5.0    32.5
  2.5mM dNTP            5.0    32.5
  2.5mM dNTP            5.0    32.5
Line 58: Line 58:
**490ng for L5 reaction: 490/30=16.3uL
**490ng for L5 reaction: 490/30=16.3uL
**500ng for L6 reaction: 500/30=16.6uL
**500ng for L6 reaction: 500/30=16.6uL
(Vortex the DNA mixture and store the sample in -20C)
(Vortex the DNA mixture and store the sample in -20C)
*Elute DNA in pre-warmed PB (20 + 8uL)


===A-tailing===
===A-tailing===
Line 67: Line 67:


                       1rxn    6.5rxn
                       1rxn    6.5rxn
  DNA                   28.0      -
  DNA+H2O              28.0      -
  10X buf.              4.0    26.0
  10X buf.              4.0    26.0
  1mM dATP              6.0    39.0
  1mM dATP              6.0    39.0

Revision as of 02:33, 22 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-repairing recipe).
    • Get the reference position of post-ligation reactions from internal marker template.
    • Compare the effect of adding De-P-carrier or not on library construction.
    • Test the effect of contamination by 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

NOTE: reactions L1, L2 and L3 will be added with De-P-carrier. Reactions L4, L5 and L6 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
  • 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.

End-reparing & Phosphorylation

  • End-it Kit (EPICENTRE)
    • Set up reactions on cold block
                      1rxn     6.5rxn
DNA+H2O               34.0      -
10X buf.               5.0     32.5
2.5mM dNTP             5.0     32.5
10mM  ATP              5.0     32.5
Enzyme                 1.0      6.5
------------------------------------
                      50.0    104.0   104/6.5=16 --- DNA 34uL
  • Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing)
  • No inactivation needed, but purify the blunted DNA with QIAquick column.
  • After 45min incubation, add the product (50uL) into 1.5-mL non-sticky tube containg 250uL PB (QIAquick buffer)
  • Adding De-P-Carrier into the DNA mixture
    • Use De-P-Carrier "3"(100bp): 22uL*40ng/uL/30uL = 30ng/uL
    • 450ng for L4 reaction: 450/30=15.0uL
    • 490ng for L5 reaction: 490/30=16.3uL
    • 500ng for L6 reaction: 500/30=16.6uL

(Vortex the DNA mixture and store the sample in -20C)

  • Elute DNA in pre-warmed PB (20 + 8uL)

A-tailing

  • Exo-minus klenow DNA pol. kit (Epicentre)
    • Dilute the 100mM dATP to 1mM dATP
    • Set up reactions on cold block
                      1rxn     6.5rxn
DNA+H2O               28.0      -
10X buf.               4.0     26.0
1mM dATP               6.0     39.0
Enzyme                 2.0     13.0
------------------------------------
                      40.0     78.0   78/6.5=12 --- DNA 28uL