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

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**Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
**Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
**Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
**Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
  [[File:Example.jpg]]
  [[File:sam022210-2nd PCR lib.JPG|400px]]

Revision as of 22:18, 25 February 2010

Low-Input DNA libarary construction - Internal marker templates test-1

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
  • Size-selection 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                           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. Air dry the pellet. Dissolve the pellet in 34uL H2O.

End-reparing & Phosphorylation

  • End-it Kit (EPICENTRE)
                      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 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
  • Adding De-P-Carrier into the DNA mixture during QIAquick washing.
    • 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
  • Elute DNA in 28uL EB

A-tailing

  • Exo-minus klenow DNA pol. kit (Epicentre)
                      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 
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
  • QIAquick purification. Eluted in 20uL EB.

ligation

  • T4 ligation kit (Enzymatic)
                      1rxn     6.5rxn
DNA+H2O               11.0      -
2X rapid buf.         15.0     97.5
20uM Y-adaptor         2.0     13.0 
T4 ligase              2.0     13.0
------------------------------------
                      30.0    123.5/6.5=19 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • QIAquick purification. Eluted in 20uL EB.

Libraray amplification by qPCR

  • 2X Fusion mastermix (Enzymatic)
                      1rxn     6.5rxn
H2O                    0.8      5.2
DNA                   20.0      -
PCR_f (10uM)           2.0     13.0 
PCR_r (10uM)           2.0     13.0
SYBR 50X               0.2      1.3
2X Fusion enzyme      25.0    162.5
------------------------------------
                      50.0    195.0/6.5=30 
  • The amplification was stopped at the 11th cycle.

TBE-PAGE validation

File:Sam021710-qPCR 11C-new.jpg
  L1          L2          L3           L4             L5            L6
  200ng DNA   50ng DNA    10ng DNA     50ng DNA       10ng DNA      0ng DNA
  300ng tRNA  450ng tRNA  490ng tRNA   450ng tRNA     490ng tRNA    500ng tRNA
                                       450ng Carrier  490ng Carrier 500ng Carrier
  • DNA size(200bp), carrier size(100bp), Y-adaptor(PE) size(100bp)
  • All amplicons showed smear since the templates were non-size selected DNA
    • Although the theoretically only adaptor ligated DNA should be amplified, the results showed overall unspecific amplification especially in carrier-added tests (L4, L5 and L6).
    • It is also possible that the over-amplification cause these unspecific amplification (The reaction was stopped at 11th cycle for the current test). However, insufficient amplification won't get enough product for low-template DNA reaction such as starting DNA< 10ng.
  • The non-carrier test (L1, L2 and L3) showed potential targets band at expected size (~300bp) with gradient intensity. Even the 10ng DNA test (L3) can show the band with 5uL amplified PCR amplicons.
  • The carrier-added tests showed weaker potential targets band.
  • It is possible the carrier was ligated with Y-adaptors, which generate the band at size of ~200bp.
    • I should add the Heat-inactivation after I did the End-repairing reaction.

Size selection

  • 20uL PCR product samples were run on 5-well PAGE (200V, 30min).
    • Sample well: 20uL sample + 10uL 6X loading dye + 10uL 0.5X TBE buffer
    • Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer
File:Sam022210-pre size selection -L1-L2-L3.jpg  File:Sam022210-post size selection -L1-L2-L3.jpg

File:Sam022210-pre size selection -L4-L5-L6-new.jpg  File:Sam022210-post size selection -L4-L5-L6.jpg
  • DNA was recovered from the gel slice and purified using EtOH purification.
  • Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.

2nd Libraray amplification by qPCR

  • 2X Fusion mastermix (Enzymatic)
                      1rxn     6.5rxn
H2O                   10.8     70.2
DNA                   10.0      -
PCR_f (10uM)           2.0     13.0 
PCR_r (10uM)           2.0     13.0
SYBR 50X               0.2      1.3
2X Fusion enzyme      25.0    162.5
------------------------------------
                      50.0    195.0/6.5=30 
  • The amplification was stopped at the 7th cycle.

TBE-PAGE validation

  • 3uL PCR products were run on 10-well PAGE (200V, 30min).
    • Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
    • Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
File:Sam022210-2nd PCR lib.JPG