Sam:LabNotes/Microbiome-new/2010-3-2: Difference between revisions

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===End-repairing & Phosphorylation===
==End-reparing & Phosphorylation===
*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.  
Line 69: Line 63:
===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.  
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===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:samXXXX- B1 to B4 pre-cut.jpg|400px]]  [[File:samXXXX- B1 to B4 post-cut.jpg|400px]]
   
   
  [[File:sam022810- B5 to B8 pre-cut.jpg|400px]]  [[File:sam022810- B5 to B8 post-cut.jpg|400px]]
  [[File:samXXXX- B5 to B8 pre-cut.jpg|400px]]  [[File:samXXXX- 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
**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.
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                       50.0    195.0/6.5=30  
                       50.0    195.0/6.5=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 th cycle and th cycle.


===TBE-PAGE validation===
===TBE-PAGE validation===
Line 120: Line 113:
**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:samXXXX.JPG|320px]]  [[File:samXXXX.JPG|300px]]
  '''The library from B5 didn't showed up in 8th cycle due to the insufficinet amplification'''.  
  '''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'''.
**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'''.

Revision as of 01:22, 5 March 2010

Single-bacterium DNA library construction - test 1

Background

  • 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

  • Prepare the following samples:
    • DNAse digested-cleaned (D-C) tRNA 83ng/uL
    • Ecoli DNA (EDNA)
    • Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)
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 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 DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.


End-reparing & Phosphorylation=

  • 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

  • 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

  • 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

  • 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:SamXXXX- B1 to B4 pre-cut.jpg  File:SamXXXX- B1 to B4 post-cut.jpg

File:SamXXXX- B5 to B8 pre-cut.jpg  File:SamXXXX- B5 to B8 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

  • 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 
  • Only use 10uL (out of 20uL) template.
  • The amplification was stopped at the th cycle and th 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:SamXXXX.JPG  File:SamXXXX.JPG
The library from B5 didn't showed up in 8th cycle due to the insufficinet amplification. 

Discussion