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

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>Sam Chiang
(New page: ='''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 prep...)
 
>Sam Chiang
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Line 36: Line 36:


==A-tailing==
==A-tailing==
*Exo-Minus Klenow DNA polymerase(EPICENTRE)
*Comparing two Klenow(-) enzymes
*Klenow fragment (3'->5' exo-)(NEB)
**Exo-Minus Klenow DNA polymerase(EPICENTRE) - for A1, A3, A5, A7
**Klenow fragment (3'->5' exo-)(NEB) - for A2, A4, A6, A8
              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


*Exp. design


  A1  A2  A3  A4  A5  A6 ("A" tailing)
               1rxn    4.5rxn(NEB)      4.5rxn(EPI)
  NEB  EPI  NEB  EPI  EPI  EPI
               1rxn    2.5rxn(NEB)      4.5rxn(EPI)
  Blunted DNA  20
  Blunted DNA  20
  H2O            8      20                36
  H2O            8      20                36

Revision as of 18:00, 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)
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
              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)      4.5rxn(EPI)
Blunted DNA   20
H2O            8      20                36
10X buf.       4      10(10X buf."2")   18 (10X Knenow buf.)
1mM dATP       6      15                27
Enzyme         2       5                 9
------------------------------------------
              40      50/2.5=20         90/4.5=20 --- DNA 20uL
  • Transfer 20uL blunted DNA into PCR tubes
  • Transfer 20uL master mix into reactions
  • Mix the reaction by vortexing 5sec. Incubate at 37 for 30min -> 75C 20min (Inactivation)
  • Purify the A-tailed DNA with QIAquick column. Elute in 20uL H2O.

Ligation

  • T4 ligation kit (Enzymatic)
  • Only try the positive control in two different sources of Y-adaptor (Sam-made or Jie-made)
  • Adaptor:Insert calculation
    • The original positive control DNA is 358 ng/uL by Nanodrop(H2O blank)
    • Assume we use 358ng (1uL) in the begining. After two QIAquick washing => 250ng/rxn
    • The sample was seperated into two => 125ng/rxn. If the sample is around 150bp then 125/(660x150)x10^6=2525nM --- Insert
    • By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
  • Exp. design
    • A5-1, A5-2, A6-1, A6-2 (A5-1 and A6-1 using Y-adaprot from Sam; A5-2 and A6-2 using Y-adaprot from Jie)
                             2.5rxn 
 DNA                    10    -
 2X rapid liagas buf.   15   37.5
 T4 ligase               2    5.0
 20uM Y-adaptor          3    7.5
 --------------------------------
                       30   50.0/2.5=20uL --- DNA 10 uL

 Incubate at RT for 15min. Immediately followed by QIAquick purification. Elute in 20uL H2O

PAGE TBU gel evaluation

  • Sample denaturing: Mixed in PCR tube -> 75C 10min -> immediately put on ice for 1min
    • Ladder: 6uL Low mass(prediluted) + 3uL 6X loading dye.
    • Sample: 3uL sample + 3uL 0.5X TBE + 3uL 6X loading buf.

Results

File:Sam102309-TBU.jpg File:Sam102309-TBE.jpg
From left to right: 5-1, 5-2, 6-1, 6-2
  • The TBU is not successful since the sample flow out during the loading. The low mass ladder is strange for the position of 100bp band. There shouldn't be a huge seperation between 100bp and 200bp bands.
  • I ran the same samples on TBE gel. A very dim band could be found above 400bp band (difficult to be seen on the picture). If Alice's assumption is right that Y-adaptor ligated DNA will be hampered during gel electrophoresis migration, then these dim bands might be our target (expected size: 150bp + 100bp (Y-adaptor) = 250bp
File:Sam102509-TBU-correct.jpg
 From left to right: 5-1, 5-2, 6-1, 6-2
  • A new TBU-PAGE was run with corrected procedures
    • 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
    • Load denatured samples and ladders(20uL). Run at 200V for 30min.
    • Stained with 3uL SYBR-Gold in TBE for 5min
  • With a longer exposure time, a smear band could be found around 225~250bp. It is possible the ligated insert. Unfortunately, the band is weak to be cut for qPCR testing.
  • I noticed that the weak band could be cuased by the low concentration of ligation product.
    • Original amount: 358ng/rxn
    • Estimated amount before ligation(50%): 179ng/rxn
    • Divided into two for two ligations: 89.5ng/rxn
    • Ligation result 89.5ng/30uL => 2.98 ng/uL
    • TBU loading 10uL => 30ng/well