Sam:LabNotes/Human/2009-10-29

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Solexa sequencing library construction - real samples (human chro MDA product)[edit]

Background[edit]

  • Sample information
    • Sample No.10: 1/12.5 (200chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
    • Sample No.12: 1/25 (100chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
  • Only 10uL of each sample were used for this construction (save another 10uL sample as backup).

TBE-PAGE size selection[edit]

  • TBE-PAGE condition: 5-well TBU. Run at 200V, 30min
    • Sample: 10uL sample + 5uL H2O + 5uL 6X loading dye
    • Ladder: 0.5uL 25bp ladder + 15uL H2O + 5uL 6X loading dye
    • Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
  • Cut the size 250+/-50bp (200bp~300bp)
File:Sam102909-TBE-size selection.JPG File:Sam102909-TBE-size selection-post cutting.JPG
          Left: Pre-cutting                              Right: Post-cutting

Blunting[edit]

  • End-it Kit (EPICENTRE)
    • Set reactions on cold block
                      1rxn     2.5rxn
DNA                   28.0      -
10X blunting buf.      4.0     10.0
dNTP mix               4.0     10.0
Blunting Enzyme        4.0     10.0
------------------------------------
                      40.0     30.0   30/2.5=12 --- DNA 28uL
  • Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing)
  • No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.

A-tailing[edit]

  • Exo-Minus Klenow DNA polymerase(EPICENTRE)
    • Set reactions on cold block
  • Prepare the fresh dilution of 100mM dATP to 1mM dATP
              1rxn    2.5rxns(EPI)
DNA           30        -
10X buf.       4      10 (10X Knenow buf.)
1mM dATP       6      15
Klenow enzyme  2       5
------------------------------------------
              40      30/2.5=12 - DNA 30uL
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation)
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.

Ligation[edit]

  • T4 ligation kit (Enzymatic)
    • Set reactions on cold block
  • Adaptor:Insert calculation
    • Assume that only 25% DNA left after all of previous steps. 360ng*25% =90ng
    • DNA before ligation: 90ng of 250bp
    • 90/(660x250)x10^6/1000 = 0.54uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 5.4uM ~ 27uM Y-adaptor needed.
    • I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
                              2.5rxn 
 A-tailed DNA           11     -
 2X rapid liagas buf.   15    37.5
 20uM Y-adaptor          2     5.0
 T4 ligase               2     5.0
 ---------------------------------
                        30    47.5/2.5 = 19uL --- DNA 11 uL

  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB.

TBU-PAGE size selection[edit]

  • TBU-PAGE condition: 5-well TBU. Run at 200V, 30min
    • Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
    • Ladder: 0.5uL 25bp ladder + 9uL H2O + 10uL 2X TBU-loading dye
    • Heat denaturing: 75C 15min -> 1min on cool block
    • Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam103009-TBU.jpg File:Sam103009-TBU-post cutting.jpg
Left: Pre-cutting                                        Right: Post-cutting

Size selected DNA recovery from TBU-PAGE[edit]

  • Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube.
  • Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C for 2hr on the shaker. shaking at lowest speed.
  • Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant on a Nanosep column filter.
  • Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
  • Perform DNA purification with EtOH/NaOAc method.
    • Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue. Incubate at -80C for 25min (or longer)
    • Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant.
    • Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
    • Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.