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>Sam Chiang
(Created page with '='''Single-bacterium DNA library construction - test 1'''= ==Background== *Both of following protocols were working for low-input DNA library construction: **Size selection prio…')
 
>Sam Chiang
 
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==Background==
==Background==
*Both of following protocols were working for low-input DNA library construction:
*Both of following protocols were working for low-input DNA library construction:
**Size selection prior PCR amplification (with/without De-P-Carrier)  
**Size selection '''prior PCR amplification''' (with/without De-P-Carrier)  
**Size selection after 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.
*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.  
*This experiment also includes the tests of different amount of positive control using sheared DNA.


==Procedures==
==Procedures==
*Prepare the following samples
*Prepare the following samples:
**E.coli internal marker (IM) (200bp): 43.6ng/uL
**DNAse digested-cleaned (D-C) tRNA 83ng/uL
**1/10 diluted E.coli internal marker (IM) (200bp): 4.36ng/uL
**Ecoli DNA (EDNA)(Covaris sheared ~200bp) http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-2-4
**DNAse digested-cleaned (D-C) tRNA 109 ng/uL
**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    '''
                                  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.


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
**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===
*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]]


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


===Libraray amplification by qPCR===
===Libraray amplification by qPCR===
*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
  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'''.
*The amplification was stopped at the 7th 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:sam022210-2nd PCR lib.JPG|400px]]
  [[File:sam030510-lib amp 8th cycle.jpg|320px]]  [[File:sam030510-lib amp 10th cycle.JPG|300px]]
              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==
==Discussion==
*The the Dephosphorylated carriers are still ligated with adaptor in this tests.
*All libraires were working as we expected.
**Sol1: Adjust the dephosphorylation conditions.
*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.
**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.
*The libraries C4 and C5 (from single cell genome 11-1 and 12-1, respectively) were ready for sequencing.
**'''I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.'''
**16S-PCR info: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-28
*In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.
**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.  
**We hold the sequencing of the C4 and C5 librery.

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.