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*Prepare the following samples:
*Prepare the following samples:
**DNAse digested-cleaned (D-C) tRNA 83ng/uL
**DNAse digested-cleaned (D-C) tRNA 83ng/uL
**Ecoli DNA (EDNA)
**Ecoli DNA (EDNA)(Covaris sheared ~200bp) http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-2-4
**Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)
**Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)(Covaris sheared ~200bp)
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-27


  Sample name'''                        C1            C2          C3            C4            C5            C6    '''
  Sample name'''                        C1            C2          C3            C4            C5            C6    '''
Line 19: Line 20:
  --------------------------------------------------------------------------------------------------------------------
  --------------------------------------------------------------------------------------------------------------------
  Sheared DNA                        50.00        50.00      50.00        50.00          50.00          -
  Sheared DNA                        50.00        50.00      50.00        50.00          50.00          -
  E.coli IM     (43.6ng/uL)             -            -          -            -              -            1.15
  E.coli Internal Marker(IM)(43.6ng/uL) -            -          -            -              -            1.15
  D-C-tRNA      (83ng/uL)            3.60          5.42        5.90        5.42          5.42          5.42
  D-C-tRNA      (83ng/uL)            3.60          5.42        5.90        5.42          5.42          5.42
  H2O                                46.40        44.58      44.10        44.58          44.58        93.43
  H2O                                46.40        44.58      44.10        44.58          44.58        93.43
  ---------------------------------------------------------------------------------------------------------------------
  ---------------------------------------------------------------------------------------------------------------------
                                     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


*Purify the DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.
*Purify the DNA mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.




===End-repairing & Phosphorylation===
===End-repairing===
*End-it Kit (EPICENTRE)  
*End-it Kit (EPICENTRE)  


                       1rxn    8.5rxn
                       1rxn    6.5rxn
  DNA+H2O              34.0      -
  DNA+H2O              34.0      -
  10X buf.              5.0    42.5
  10X buf.              5.0    32.5
  2.5mM dNTP            5.0    42.5
  2.5mM dNTP            5.0    32.5
  10mM  ATP              5.0    42.5
  10mM  ATP              5.0    32.5
  Enzyme                1.0      8.5
  Enzyme                1.0      6.5
  ------------------------------------
  ------------------------------------
                       50.0    136.0  136/8.5=16  
                       50.0    104.0  104/6.5=16  


*Mix the reaction by vortexing 5sec. Incubate at RT for 45min.  
*Mix the reaction by vortexing 5sec. Incubate at RT for 45min.  
*Adding De-P-Carrier into the DNA mixture during QIAquick washing.
*Purify the DNA with QIAquick. Elute DNA in 28uL EB
*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===
===A-tailing===
*Exo-minus klenow DNA pol. kit (Epicentre)  
*Exo-minus klenow DNA pol. kit (Epicentre)  


                       1rxn    8.5rxn
                       1rxn    6.5rxn
  DNA+H2O              28.0      -
  DNA+H2O              28.0      -
  10X buf.              4.0    34.0
  10X buf.              4.0    26.0
  1mM dATP              6.0    51.0
  1mM dATP              6.0    39.0
  Enzyme                2.0    17.0
  Enzyme                2.0    13.0
  ------------------------------------
  ------------------------------------
                       40.0   102.0  102/8.5=12  
                       40.0     78.0  78/6.5=12  


*Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.  
*Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.  
*QIAquick purification. Eluted in 20uL EB.
*QIAquick purification. Eluted in 12uL EB.
 


===ligation===
===Ligation===
*T4 ligation kit (Enzymatic)
*T4 ligation kit (Enzymatic)
                       1rxn    8.5rxn
                       1rxn    6.5rxn
  DNA+H2O              11.0      -
  DNA+H2O              11.0      -
  2X rapid buf.        15.0   127.5
  2X rapid buf.        15.0     97.5
  20uM Y-adaptor        2.0    17.0  
  20uM Y-adaptor        2.0    13.0  
  T4 ligase              2.0    17.0
  T4 ligase              2.0    13.0
  ------------------------------------
  ------------------------------------
                       30.0    161.5/8.5=19  
                       30.0    123.5/6.5=19  


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


===Size selection===
===Size selection===
*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).
*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
**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
**Ladder well: 1.0uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer


  [[File:sam022810- B1 to B4 pre-cut.jpg|400px]]  [[File:sam022810- B1 to B4 post-cut.jpg|400px]]
  [[File:sam030410-C1 C2 C3.jpg|350px]]  [[File:sam030410-C1 C2 C3 - post cut.jpg|400px]]
   
   
  [[File:sam022810- B5 to B8 pre-cut.jpg|400px]]  [[File:sam022810- B5 to B8 post-cut.jpg|400px]]
  [[File:sam030410-C4 C5  C6.jpg|350px]]  [[File:sam030410-C4 C5  C6 - post cut.jpg|350px]]
'''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
**During the 37C incubation step (1X TE and homogenized gel slides), additional tRNA carriers were added into each reaction to protect sample DNA.
**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'''
  Sample name'''                       C1           C2           C3           C4           C5            C6    '''
                                   200ng IM        50ng IM      10ng IM      50ng IM      10ng IM        0ng IM        0ng IM        0ng IM
                                   200ng EDNA      50ng EDNA    10ng EDNA    50ng P11-1    50ng P12-1    50ng IM  
                                   300ng tRNA    450ng tRNA  490ng tRNA    450ng tRNA    490ng tRNA   500ng tRNA    500ng tRNA    500ng tRNA
                                   300ng tRNA    450ng tRNA  490ng tRNA    450ng tRNA    450ng tRNA     450ng tRNA  
  ---------------------------------------------------------------------------------------------------------------------------------------------
  --------------------------------------------------------------------------------------------------------------------
  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       (83ng/uL)             3.60          5.42        5.90         5.42          5.42         5.42 uL
 
*Incubate the gel mixture with '''500uL 1X TE at 37C for 2hr'''.
*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.


===Libraray amplification by qPCR===
===Libraray amplification by qPCR===
Line 110: Line 102:
  PCR_r (10uM)          2.0    13.0
  PCR_r (10uM)          2.0    13.0
  SYBR 50X              0.2      1.3
  SYBR 50X              0.2      1.3
  2X Fusion enzyme     25.0    162.5
  2X Phusion HF enzyme 25.0    162.5
  ------------------------------------
  ------------------------------------
                       50.0    195.0/6.5=30  
                       50.0    260.0/6.5=30  
*'''Only use 10uL (out of 20uL) template'''.  
*'''Only use 10uL (out of 20uL) template'''.  
*The amplification was stopped at the 8th cycle and 12th cycle.
*The amplification was stopped at the 8th cycle and 10th cycle.


===TBE-PAGE validation===
===TBE-PAGE validation===
Line 120: Line 112:
**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:sam030210-PCR amplified lib - 8th cycle.JPG|320px]]  [[File:sam030210-PCR amplified lib - 12th cycle.JPG|300px]]
  [[File:sam030510-lib amp 8th cycle.jpg|320px]]  [[File:sam030510-lib amp 10th cycle.JPG|300px]]
'''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==
==Discussion==
*'''Regardless which enzyme or condition was used, all dephosphorylated carriers are still ligated with adaptor in this test'''.
*All libraires were working as we expected.
**Even I did the inactivation step (70C 10min) following the End-repairing step, the situation was not improved.
*For the C5 sample, '''it is unknown why 10th cycle amplification has less background than 8th cycle amplification'''. I run another gel to check them (results not shown) and results are still the same.
**B4-B6: De-P-Carrier prepared by SAP, 30min.
**B5:   De-P-Carrier prepared by SAP, 60min.
*The libraries C4 and C5 (from single cell genome 11-1 and 12-1, respectively) were ready for sequencing.
**B6:    De-P-Carrier prepared by ACP, 60min.
**16S-PCR info: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-28
*Possible reasons of the appearance of adaptor-ligated carrier:
**After discussed with Dr. Zhang, we will prefer to sequence sample from E.coli single cell genome in which we can optimized the protocol of library construction.  
**The Dephosphorylation is not complete.
**We hold the sequencing of the C4 and C5 librery.
**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 05:49, 10 July 2011

Single-bacterium DNA library construction - test 1[edit]

Background[edit]

  • Both of following protocols were working for low-input DNA library construction:
    • Size selection prior PCR amplification (with/without De-P-Carrier)
    • Size selection after PCR amplification (with/without De-P-Carrier)
  • I decided to use the method with size selection prior PCR amplification, but without adding De-P-Carriers.
  • This experiment also includes the tests of different amount of positive control using sheared DNA.

Procedures[edit]

Sample name                        C1            C2           C3            C4            C5             C6     
                                 200ng EDNA      50ng EDNA    10ng EDNA     50ng P11-1    50ng P12-1     50ng IM 
                                 300ng tRNA     450ng tRNA   490ng tRNA    450ng tRNA    450ng tRNA     450ng tRNA   
--------------------------------------------------------------------------------------------------------------------
Sheared DNA                         50.00         50.00       50.00        50.00          50.00           -
E.coli Internal Marker(IM)(43.6ng/uL)  -            -           -            -              -            1.15
D-C-tRNA       (83ng/uL)             3.60          5.42        5.90         5.42           5.42          5.42
H2O                                 46.40         44.58       44.10        44.58          44.58         93.43
---------------------------------------------------------------------------------------------------------------------
                                   100.00        100.00      100.00       100.00         100.00        100.00 uL
  • Purify the DNA mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.


End-repairing[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.
  • Purify the DNA with QIAquick. 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 12uL 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.


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: 1.0uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer
File:Sam030410-C1 C2 C3.jpg  File:Sam030410-C1 C2 C3 - post cut.jpg

File:Sam030410-C4 C5 C6.jpg  File:Sam030410-C4 C5 C6 - post cut.jpg
  • 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                        C1            C2           C3            C4            C5             C6     
                                 200ng EDNA      50ng EDNA    10ng EDNA     50ng P11-1    50ng P12-1     50ng IM 
                                 300ng tRNA     450ng tRNA   490ng tRNA    450ng tRNA    450ng tRNA     450ng tRNA   
--------------------------------------------------------------------------------------------------------------------
D-C-tRNA       (83ng/uL)             3.60          5.42        5.90         5.42           5.42          5.42 uL
  • Incubate the gel mixture with 500uL 1X TE at 37C for 2hr.
  • 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 Phusion HF enzyme  25.0    162.5
------------------------------------
                      50.0    260.0/6.5=30 
  • Only use 10uL (out of 20uL) template.
  • The amplification was stopped at the 8th cycle and 10th 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:Sam030510-lib amp 8th cycle.jpg  File:Sam030510-lib amp 10th cycle.JPG

Discussion[edit]

  • All libraires were working as we expected.
  • For the C5 sample, it is unknown why 10th cycle amplification has less background than 8th cycle amplification. I run another gel to check them (results not shown) and results are still the same.