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

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Revision as of 19:36, 5 March 2010 by >Sam Chiang (→‎Libraray amplification by qPCR)
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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-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: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

  • 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    260.0/6.5=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