Sam:LabNotes/Microbiome-new/2010-3-2

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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-repairing & Phosphorylation

  • 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

  • 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

  • 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

  • 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

  • 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 8th cycle and 12th 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: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

  • 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.