Sam:LabNotes/Human/2009-10-25: Difference between revisions

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  [[Image:sam102609-TBU-4th test.jpg|450px]] [[Image:sam102609-TBU-4th test -post cut.jpg|450px]]
  [[Image:sam102609-TBU-4th test.jpg|450px]] [[Image:sam102609-TBU-4th test -post cut.jpg|450px]]
  Left: Pre-cutting                           Right: Post-cutting
  Left: Pre-cutting                                         Right: Post-cutting
   
   
  NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting.
  NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting.

Revision as of 23:32, 29 October 2009

Solexa sequencing library construction - 4th test

Background

  • I noticed that I can use human chromosome primer to generate the PCR product for positive control.

Sample preparation

  • Prepare two types of postive control with human chro primer P1(270bp) and P11 (130bp)
    • Template: Jurket gDNA
    • Purify the PCR product using Montage filter (Millipore). Elute in 20uL H2O.
    • Results: P1 DNA(270bp): 158.16 ng/uL in 20uL. P11 DNA(130bp): 92.82 ng/uL in 20uL.

Blunting

  • Set up 8 reaction with 400ng DNA for starting amount. Add H2O to DNA sample to make the volume needed for reaction.
  • Comparing End-it kit(EPI) and Quick blunting kit(NEB)
    • Set reactions on cold block
End-it Kit - test on sample B1, B2, B3, B4
                      1rxn     4.5rxn
DNA                   28.0      -
10X blunting buf.      4.0     18.0
1mM dNTP mix           4.0     18.0
Blunting Enzyme        4.0     18.0
------------------------------------
                      40.0     54.0   54/4.5=12 --- DNA 28uL
Quick blunting Kit - test on B5, B6, B7, B8
                      1rxn     4.5rxn
DNA                   18.0      -
10X blunting buf.      2.5     11.25
1mM dNTP mix           2.5     11.25
Blunting Enzyme        2.0      9.0
------------------------------------
                      25.0     31.5   31.5/4.5=7 --- DNA 18L 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
  • No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.


A-tailing

  • Comparing two Klenow(-) enzymes
    • Exo-Minus Klenow DNA polymerase(EPICENTRE) - for A1, A3, A5, A7
    • Klenow fragment (3'->5' exo-)(NEB) - for A2, A4, A6, A8
    • Set reactions on cold block
              1rxn     4.5rxns(EPI)
Blunted DNA   30        -
10X buf.       4      18 (10X Knenow buf.)
1mM dATP       6      27
Enzyme         2       9
------------------------------------------
              40      54/4.5=12 - DNA 30uL


              1rxn    4.5rxn(NEB) 
Blunted DNA   28
10X buf.       4      18(10X buf."2")  
1mM dATP       6      27               
Enzyme         2       9
------------------------------------------
              40      54/4.5=12 - DNA 28uL
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.


Ligation

  • T4 ligation kit (Enzymatic)
  • Only use the Y-adaprot I made (1st made Y-adaptor)
  • Adaptor:Insert calculation
    • Assume that there is only 50% DNA left after all of prevous steps (Note: There is no size-selection in this test).
    • DNA before ligation: 200ng of 270bp and 200ng of 130bp (Starting amount: 400ng)
    • 200/(660x270)x10^6= 1122.33nM = 1.122uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 11.23uM ~ 56.11uM Y-adaptor needed.
    • By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
    • 200/(660x130)x10^6= 2331.00nM = 2.331uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 23.31uM ~ 116.55uM Y-adaptor needed.
    • By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
                              9rxn 
 A-tailed DNA           10     -
 2X rapid liagas buf.   15   135.0
 20uM Y-adaptor          3    27.0
 T4 ligase               2    18.0
 ---------------------------------
                        30   180.0/9=20uL --- DNA 10 uL

  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 10uL EB (All of 10uL ligation will used for TBU-PAGE size selection).

PAGE TBU gel size selection

  • A 5-well TBU gel is recommanded since it has large capacity for sample loading.
    • 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
    • Flush TBU gell well, pre-run 10min at 200V
    • Flush TBU gell well. Load denatured samples and ladders(20uL). Run at 200V for 30min.
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam102609-TBU-4th test.jpg File:Sam102609-TBU-4th test -post cut.jpg
Left: Pre-cutting                                          Right: Post-cutting

NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting.