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

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Revision as of 19:21, 22 February 2010 by >Sam Chiang
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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 immediately.
  • 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
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min (Program "A-tailing").
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation).
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column immediately. Eluted in pre-warmed EB (10 + 10uL).
  • Speed vac the sample from 20uL to 11uL (around 10min at default speed).
  • NOTE: During the A-tailing step. Prepare the Y-adaptor annealing.
  • Preparign 20uM Y-adaptor mix
  100uM Sam-PE_t      20uL
  100uM Sam-PE_b      20uL
  10X T4 ligase buf.  10uL
  H2O                 50uL
  ------------------------
                     100uL
  94C 3min -> cool donw to 20C at the rate of 0.1/sec using RAMP function

TA 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 --- newly synthesized Sam-PE-adaptor (20uM)
T4 ligase              2.0     13.0
------------------------------------
                      30.0    123.5/6.5=19 --- DNA 11uL
  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (library amplification by qPCR).
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column immediately. Eluted in pre-warmed EB (10+10uL).

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 --- DNA 20uL (Use all of ligation reaction from the last step)
  • Monitor the amplification curves and stop the reaction before reaching the plateau.
    • The amplification was stopped at the 11th cycle.
  • NOTE: Since the PCR amplification was performed on the non-size selected template, there are lots of noise interfere the correct timing to stop the amplification.

TBE-PAGE validation

  • Use 5uL of amplified product for validation on 10-well TBE-PAGE
    • Sample loading: 5uL sample + 5uL 0.5X TBE + 5uL 6X loading dye
    • Ladder loading: 0.5 low mass ladder + 10uL 0.5X TBE + 5uL 6X loading dye
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

  • The potential target bands were size selected on 5-well TBE-PAGE (x2).
    • Sample loading: 20uL sample + 5uL 0.5X TBE + 5uL 6X loading dye
    • Ladder loading: 0.5 low mass ladder + 20uL 0.5X TBE + 5uL 6X loading dye
    • Load ladder between each samples. Run at 200V for 30min.
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.
    • Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube.
    • Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C for 1hr on the shaker. shaking at lowest speed.
    • Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant (300~320uL on a Nanosep column filter.
    • Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
    • Sample(300uL) + 2.5V 100% EtOH(750uL) + 0.1V 3M NaOAc(30uL)+ 1.5uL Glycoblue. Incubate at -80C for 25min.
    • Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant.
    • Pellet washing with 750uL 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
    • Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.