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
    • E.coli internal marker (IM) (200bp): 43.6ng/uL
    • 1/10 diluted 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
    • 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-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
  • 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                    0.8     70.2
DNA                   20.0      -
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 
  • The amplification was stopped at the 7th 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:Sam022210-2nd PCR lib.JPG
              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

  • The the Dephosphorylated carriers are still ligated with adaptor in this tests.
    • Sol1: Adjust the dephosphorylation conditions.
    • 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.
    • I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.
  • In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.