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

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='''Low-Input DNA libarary construction - Internal marker templates test-2'''=
='''Low-Input DNA library construction - Internal marker templates test-2'''=


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


==Pocedures==
==Procedures==
*Prepare the following samples
*Prepare the following samples
**E.coli internal marker (IM) (200bp): 43.6ng/uL
**E.coli internal marker (IM) (200bp): 43.6ng/uL
Line 25: Line 25:
   
   
  '''NOTE: reactions B1, B2 and B3 will be added with De-P-carrier. Reactions B4 - B8 won't.'''
  '''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
*Purify the DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.
**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-repairing & Phosphorylation===
*End-it Kit (EPICENTRE)  
*End-it Kit (EPICENTRE)  


Line 89: Line 87:
   
   
  [[File:sam022810- B5 to B8 pre-cut.jpg|400px]]  [[File:sam022810- B5 to B8 post-cut.jpg|400px]]
  [[File:sam022810- B5 to B8 pre-cut.jpg|400px]]  [[File:sam022810- B5 to B8 post-cut.jpg|400px]]
'''The dual bands of ligation reactions suggested that some DNA were only ligated with one end but not both ends'''


*DNA was recovered from the gel slice  
*DNA was recovered from the gel slice  
Line 98: Line 97:
  D-C-tRNA(78ng/uL)                  3.85          5.77        6.28          5.77          6.28          6.41          6.41          6.41 uL
  D-C-tRNA(78ng/uL)                  3.85          5.77        6.28          5.77          6.28          6.41          6.41          6.41 uL
   
   
**Purify the DAN with Nanosap filter and EtOH precipitation.
*Purify the DAN with Nanosap filter and EtOH precipitation.
**Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
**Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.


Line 104: Line 103:
*2X Fusion mastermix (Enzymatic)
*2X Fusion mastermix (Enzymatic)
                       1rxn    6.5rxn
                       1rxn    6.5rxn
  H2O                   0.8    70.2
  H2O                   10.8    70.2
  DNA                  20.0      -
  DNA                  10.0      -
  PCR_f (10uM)          2.0    13.0  
  PCR_f (10uM)          2.0    13.0  
  PCR_r (10uM)          2.0    13.0
  PCR_r (10uM)          2.0    13.0
Line 111: Line 110:
  2X Fusion enzyme      25.0    162.5
  2X Fusion enzyme      25.0    162.5
  ------------------------------------
  ------------------------------------
                       50.0    195.0/6.5=30
                       50.0    260.0/6.5=40
 
*'''Only use 10uL (out of 20uL) template'''.
*The amplification was stopped at the 7th cycle.
*The amplification was stopped at the 8th cycle and 12th cycle.


===TBE-PAGE validation===
===TBE-PAGE validation===
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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:sam022210-2nd PCR lib.JPG|400px]]
  [[File:sam030210-PCR amplified lib - 8th cycle.JPG|320px]] [[File:sam030210-PCR amplified lib - 12th cycle.JPG|300px]]
               L1     L2     L3       L4       L5       L6'''
'''The library from B5 didn't showed up in 8th cycle due to the insufficinet amplification'''.
               B1     B2     B3       B4       B5       B6'''
  Input DNA    200ng  50ng  10ng    50ng    10ng    0ng  
  Input DNA    200ng  50ng  10ng    50ng    10ng    0ng  
  tRNA          300ng  450ng 490ng    450ng    490ng  500ng  
  tRNA          300ng  450ng 490ng    450ng    490ng  500ng  
Line 133: Line 134:


==Discussion==
==Discussion==
*The the Dephosphorylated carriers are still ligated with adaptor in this tests.
*'''Regardless which enzyme or condition was used, all dephosphorylated carriers are still ligated with adaptor in this test'''.
**Sol1: Adjust the dephosphorylation conditions.
**Even I did the inactivation step (70C 10min) following the End-repairing step, the situation was not improved.
**Sol2: Add heat inactivation stet right after End-reparing reaction.
**B4-B6: De-P-Carrier prepared by SAP, 30min.
*The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples.
**B5:    De-P-Carrier prepared by SAP, 60min.
**'''I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.'''
**B6:   De-P-Carrier prepared by ACP, 60min.
*In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.
*Possible reasons of the appearance of adaptor-ligated carrier:
**The Dephosphorylation is not complete.
**The De-P-Carriere was rephosphorylated after End-repairing.
**The Phosphorylation "on templates (insert)" is not playing the critical roll for ligation.
 
*The early size selection method is able to construct the library with only 10ng DNA (for both with or without carrier conditions).  
*The early size selection method can generate libraries '''with much less unspecific amplification'''.

Latest revision as of 06:38, 4 April 2010

Low-Input DNA library construction - Internal marker templates test-2[edit]

Background[edit]

  • Repeat the low-input DNA library construction with following changes:
    • Add inactivation step following End-repairing.
    • 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.

Procedures[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                          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. Air dry the pellet. Dissolve the pellet in 34uL H2O.

End-repairing & Phosphorylation[edit]

  • 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[edit]

  • 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.


ligation[edit]

  • T4 ligation kit (Enzymatic)
                      1rxn     8.5rxn
DNA+H2O               11.0      -
2X rapid buf.         15.0    127.5
20uM Y-adaptor         2.0     17.0 
T4 ligase              2.0     17.0
------------------------------------
                      30.0    161.5/8.5=19 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • QIAquick purification. Eluted in 20uL EB.

Size selection[edit]

  • Only size select the first six reactions (reaction B7 and B8 are two extra negative controls).
  • 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:Sam022810- B1 to B4 pre-cut.jpg  File:Sam022810- B1 to B4 post-cut.jpg

File:Sam022810- B5 to B8 pre-cut.jpg  File:Sam022810- B5 to B8 post-cut.jpg
The dual bands of ligation reactions suggested that some DNA were only ligated with one end but not both ends
  • DNA was recovered from the gel slice
    • During the 37C incubation step (1X TE and homogenized gel slides), additional tRNA carriers were added into each reaction to protect sample DNA.
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
---------------------------------------------------------------------------------------------------------------------------------------------
D-C-tRNA(78ng/uL)                   3.85           5.77         6.28          5.77          6.28          6.41          6.41          6.41 uL

  • Purify the DAN with Nanosap filter and EtOH precipitation.
    • Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.

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    260.0/6.5=40 
  • Only use 10uL (out of 20uL) template.
  • The amplification was stopped at the 8th cycle and 12th 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:Sam030210-PCR amplified lib - 8th cycle.JPG  File:Sam030210-PCR amplified lib - 12th cycle.JPG
The library from B5 didn't showed up in 8th cycle due to the insufficinet amplification. 

              B1      B2     B3       B4       B5       B6
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]

  • Regardless which enzyme or condition was used, all dephosphorylated carriers are still ligated with adaptor in this test.
    • Even I did the inactivation step (70C 10min) following the End-repairing step, the situation was not improved.
    • B4-B6: De-P-Carrier prepared by SAP, 30min.
    • B5: De-P-Carrier prepared by SAP, 60min.
    • B6: De-P-Carrier prepared by ACP, 60min.
  • Possible reasons of the appearance of adaptor-ligated carrier:
    • The Dephosphorylation is not complete.
    • The De-P-Carriere was rephosphorylated after End-repairing.
    • The Phosphorylation "on templates (insert)" is not playing the critical roll for ligation.
  • The early size selection method is able to construct the library with only 10ng DNA (for both with or without carrier conditions).
  • The early size selection method can generate libraries with much less unspecific amplification.