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

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>Sam Chiang
 
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='''Low-Input DNA libarary construction - Internal marker templates'''=
='''Low-Input DNA libarary construction - Internal marker templates test-1 '''=


==Background==
==Background==
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**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 effect of contamination by De-P-carrier
**Test the effect of contamination by De-P-carrier
*'''Size-selection prior PCR amplification'''


==Pocedures==
==Pocedures==
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  -----------------------------------------------------------------------------------------------------------------
  -----------------------------------------------------------------------------------------------------------------
  E.coli IM      (43.6ng/uL)          4.59          1.146        -          1.146            -            -
  E.coli IM      (43.6ng/uL)          4.59          1.146        -          1.146            -            -
  1/10 E.coli IM (4.36ng/uL0            -            -        2.293                       2.293          -
  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
  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
  H2O                                92.66        94.726      93.217      94.726        93.217        95.413
  --------------------------------------------------------------------------------------------------------------
  --------------------------------------------------------------------------------------------------------------
                                     100.00        100.00      100.00      100.00        100.00        100.00 uL
                                     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.'''
  '''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===
*Purify the DAN mixture with EtOH precipitation
**mimic the real precedures to remove the enzyme and buffers from WGA reactions
**To 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.
*Air dry the pellet. Dissolve the pellet in 34uL H2O.


===End-reparing & Phosphorylation===
===End-reparing & Phosphorylation===
Line 77: Line 78:


*Mix the reaction by vortexing 5sec. Incubate at RT for 15min.  
*Mix the reaction by vortexing 5sec. Incubate at RT for 15min.  
*QIAquick column immediately. Eluted in 20uL EB.
*QIAquick purification. Eluted in 20uL EB.


===Libraray amplification by qPCR===
===Libraray amplification by qPCR===
Line 93: Line 94:


===TBE-PAGE validation===
===TBE-PAGE validation===
*Evaluate 5uL of PCR product on TBE-PAGE
*Sample well: 5uL sample + 5uL 0.5X TBE + 5uL 6X loading dye
  [[File:sam021710-qPCR 11C-new.jpg|400px]]
  [[File:sam021710-qPCR 11C-new.jpg|400px]]
   '''L1          L2          L3          L4            L5            L6'''
   '''L1          L2          L3          L4            L5            L6'''
Line 109: Line 112:


===Size selection===
===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|400px]]  [[File:sam022210-post size selection -L1-L2-L3.jpg|400px]]
  [[File:sam022210-pre size selection -L1-L2-L3.jpg|400px]]  [[File:sam022210-post size selection -L1-L2-L3.jpg|400px]]
   
   
Line 114: Line 121:


*DNA was recovered from the gel slice and purified using EtOH purification.
*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.
*Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
**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.  
===2nd Libraray amplification by qPCR===
**Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
*2X Fusion mastermix (Enzymatic)
**'''Sample(300uL) + 2.5V 100% EtOH(750uL) + 0.1V 3M NaOAc(30uL)+ 1.5uL Glycoblue'''. Incubate at -80C for 25min.
                      1rxn    6.5rxn
**Pellet the DNA precipitation by centrifuging at '''4C, 10,000xg for 25min'''. Remove the supernatant.
H2O                  10.8    70.2
**Pellet washing with 750uL 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
DNA                  10.0      -
**Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
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|400px]]
              L1      L2    L3      L4      L5      L6'''
Input DNA    200ng  50ng  10ng    50ng    10ng    0ng
tRNA          300ng  450ng 490ng    450ng    490ng  500ng
De-P-Carrier                        450ng    490ng  500ng
Expected size:
Nucleotides      Prior-ligation      Post-ligation
--------------------------------------------------
tRNA                N/A                N/A
Sample (E.coli)    200bp              ~300bp
Carrier (Lambda)    100bp              ~200bp
 
==Discussion==
*The the Dephosphorylated carriers are still ligated with adaptor in this tests.
**Sol1: Adjust the dephosphorylation conditions.
**Sol2: Add heat inactivation stet right after End-reparing reaction.
*The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples.
**'''I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.'''
*In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.

Latest revision as of 06:06, 3 March 2010

Low-Input DNA libarary construction - Internal marker templates test-1 [edit]

Background[edit]

  • 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[edit]

  • 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                                 92.66         94.726      93.217       94.726         93.217        95.413
--------------------------------------------------------------------------------------------------------------
                                   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
    • To mimic the real precedures to remove the enzyme and buffers from WGA reactions.
  • Air dry the pellet. Dissolve the pellet in 34uL H2O.

End-reparing & Phosphorylation[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • Evaluate 5uL of PCR product on TBE-PAGE
  • Sample well: 5uL sample + 5uL 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[edit]

  • 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[edit]

  • 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[edit]

  • 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
              L1      L2     L3       L4       L5       L6
Input DNA     200ng   50ng   10ng     50ng     10ng     0ng 
tRNA          300ng   450ng 490ng    450ng    490ng   500ng 
De-P-Carrier                         450ng    490ng   500ng 

Expected size:
Nucleotides      Prior-ligation      Post-ligation
--------------------------------------------------
tRNA                 N/A                 N/A
Sample (E.coli)     200bp              ~300bp
Carrier (Lambda)    100bp              ~200bp

Discussion[edit]

  • The the Dephosphorylated carriers are still ligated with adaptor in this tests.
    • Sol1: Adjust the dephosphorylation conditions.
    • Sol2: Add heat inactivation stet right after End-reparing reaction.
  • The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples.
    • I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.
  • In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.