Jie:LabNotes/CpgSeq/2009-8-5: Difference between revisions

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>Jie deng
(New page: ==shotgun library construction of Parkinson's samples== ====endrepair with epicentre kit==== 100 ul DNA 15 ul 10X End-Repair Buffer 15 ul dNT...)
 
>Jie deng
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==shotgun library construction of Parkinson's samples==
==shotgun library construction of Parkinson's samples==
====endrepair with epicentre kit====
The captured DNA of samples were sent to Covaris for shearing.refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-7-1] see 1.5.2
                                          
===endrepair with enzymatic end-repair kit===
                                         x10
  100 ul DNA  
  100 ul DNA  
   15 ul 10X End-Repair Buffer
   13 ul 10X End-Repair Buffer           130
   15 ul dNTP Mix
   13 ul dNTP Mix                       130
  15 ul ATP
   4 ul End-Repair Enzyme Mix           40
   3 ul End-Repair Enzyme Mix
  130 ul Total reaction volume
  150 ul Total reaction volume
  Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.
  Incubate at room temperature for 45 minutes. Purify with minelute column.


==== A tail addition====
=== A tail addition===
                                     x8
                                     x11
   Blunt-ended DNA        10ul      80
   Blunt-ended DNA        10ul      10 each
   10X Klenow buffer      1.5ul     12
   10X Klenow buffer      1.6ul     17.6
   1mM dATP                3ul      24
   1mM dATP                3ul      33
   Klenow fragment (exo-)  1ul       8
   Klenow fragment (exo-)  1ul     11
   37C 30min, purified with MinElute columns, eluted with 20ul EB.  
   37C 30min, purified with MinElute columns, eluted with 12ul EB.  


After A tail, I did size selection for No7 and positive control, then do the adaptor ligation. <BR>After adaptor ligation, i goes to PCR directly without size selection again. <BR>However, I can only see the positive control product only.
===adaptor ligation===
 
                                                x12
So I skipped the size selection before adaptor ligation for No 1~6, and do the size selection after adaptor ligation. 
     DNA                                10ul
However, the expected band after PCR is so weak that I am not sure the targets were successfully amplified.
     2x QuickLigase buffer (enzymatic)  15ul    180
 
     20uM Adaptor oligo mix              3ul     36
====adaptor ligation====
     T4 DNA QuickLigase (enzymatic)      2ul     24
     DNA                                20ul
     10x QuickLigase buffer (enzymatic)  3ul
     20uM Adaptor oligo mix              3ul
     T4 DNA QuickLigase (enzymatic)      2ul
     Incubate at RT for 15 minutes.
     Incubate at RT for 15 minutes.
     Purified with Qiaquick columns, eluted with 12ul EB
     Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O.


====PCR====
===PCR===


     Template               15ul        
     Template               5ul        
     2x iProof mix          50ul       
     2x iProof mix          50ul       
     Solexa_PCR_up (10uM)    4ul         
     Solexa_PCR_up (10uM)    4ul         
     Solexa_PCR_lo_PE (10uM) 4ul         
     Solexa_PCR_lo_PE (10uM) 4ul         
     H2O                    30ul        
     H2O                    37ul        
     50X SYBG I            0.2ul    
     50X SYBG I            0.2ul
98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)->
72C 3min -> 15C hold.
 
[[Image:20090807_shotgun_lib_Parkinson_samples.jpg|350px]]20090807_shotgun_lib_Parkinson_samples
 
===quantification of shotgun library===
[[Image:20090810_shotgun_lib_quanti_Parkinson_No1_9_Phix.jpg|300px]]20090810_shotgun_lib_quanti_Parkinson_No1_9_Phix
 
gel quantification results:
Par_1: 6.14ng/ul x 40ul;
Par_9: 7.54ng/ul x 40ul;
PhiX lib: 7.43ng/ul (size 275-325bp)
 
Nanodrop results:
Par_1: 24.9ng/ul x 40ul;
Par_9: 21.8ng/ul x 40ul;
PhiX lib: 5.9ng/ul


[[Image:20090721_shotgun_libr_par7_positive control.jpg]]20090721_shotgun_libr_par7_positive control
===quantification with qPCR===
I dilute the PhiX lib(10nM) to 1nM, 0.5nM, 0.25nM, 0.05nM.
I dilute the Parkinson's lib with 1:10 ratio.
Syb_FP5: ATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
                                    x 14
  Template                1ul         
  2x iProof mix          25ul        350
  syb_RP7 (100uM)        0.2ul       
  Syb_FP5 (100uM)        0.2ul       
  H2O                    24ul     
  50X SYBG I            0.2ul
98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.


[[Image:20090721_shotgun_libr_par1~6.jpg]]20090721_shotgun_libr_par1~6
[[Image:20090812_PhiX_quantification.png|300px]]


==redo the shotgun library starting from PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-22)==
  [[Media:20090812_Parkinson_sample_quantification.xls|20090812_Parkinson_sample_quantification]]
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                x4      x17 
H2O                                                  50ul      200ul    3400
2x Mastermix                                        50ul      200ul    3400
AmpF6.3NH2(100uM)                                    0.2ul    0.8ul    13.6
AmpR6.3NH2(100uM)                                    0.2ul    0.8ul    13.6
50x SYBG I                                          0.4ul      1.6ul    27.2
template                                      1ul/each for purified cpg97k
Total                                              100ul      800ul   
  94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold.


I did the PCR reamplification with Amp6.3 primers with Parkinson's sample 1 and 10 with the same PCR condition.
{| border="1" cellpadding="5" cellspacing="0" align="center"
|-
| align="center" style="background:#f0f0f0;"|'''No'''
| align="center" style="background:#f0f0f0;"|'''sample '''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
|-
|Par_1||5028||12.2nM
|-
|Par_2||4735||10.2nM
|-
|Par_3||4789||9.3nM
|-
|Par_4||5171||6.6nM
|-
|Par_5||1818||6.0nM
|-
|Par_6||1947||7.8nM
|-
|Par_7||1741||6.9nM
|-
|Par_8||4977||9.6nM
|-
|Par_9||4879||5.5nM
|-
|Par_10||4526||6.6nM
|-
|}

Latest revision as of 18:08, 17 August 2009

shotgun library construction of Parkinson's samples[edit]

The captured DNA of samples were sent to Covaris for shearing.refer to LabNotes on [1] see 1.5.2

endrepair with enzymatic end-repair kit[edit]

                                       x10
100 ul DNA 
 13 ul 10X End-Repair Buffer           130
 13 ul dNTP Mix                        130
  4 ul End-Repair Enzyme Mix            40
130 ul Total reaction volume
Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.

A tail addition[edit]

                                   x11
  Blunt-ended DNA        10ul      10 each
  10X Klenow buffer      1.6ul     17.6
  1mM dATP                3ul      33
  Klenow fragment (exo-)  1ul      11
  37C 30min, purified with MinElute columns, eluted with 12ul EB. 

adaptor ligation[edit]

                                               x12
    DNA                                10ul
    2x QuickLigase buffer (enzymatic)  15ul     180
    20uM Adaptor oligo mix              3ul      36
    T4 DNA QuickLigase (enzymatic)      2ul      24
    Incubate at RT for 15 minutes.
    Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O.

PCR[edit]

   Template                5ul        
   2x iProof mix          50ul       
   Solexa_PCR_up (10uM)    4ul        
   Solexa_PCR_lo_PE (10uM) 4ul        
   H2O                     37ul       
   50X SYBG I             0.2ul
98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)-> 
72C 3min -> 15C hold.

File:20090807 shotgun lib Parkinson samples.jpg20090807_shotgun_lib_Parkinson_samples

quantification of shotgun library[edit]

File:20090810 shotgun lib quanti Parkinson No1 9 Phix.jpg20090810_shotgun_lib_quanti_Parkinson_No1_9_Phix

gel quantification results:
Par_1: 6.14ng/ul x 40ul;
Par_9: 7.54ng/ul x 40ul;
PhiX lib: 7.43ng/ul (size 275-325bp)
Nanodrop results:
Par_1: 24.9ng/ul x 40ul;
Par_9: 21.8ng/ul x 40ul;
PhiX lib: 5.9ng/ul

quantification with qPCR[edit]

I dilute the PhiX lib(10nM) to 1nM, 0.5nM, 0.25nM, 0.05nM.
I dilute the Parkinson's lib with 1:10 ratio. 
Syb_FP5: ATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
                                    x 14
  Template                 1ul          
  2x iProof mix           25ul        350
  syb_RP7 (100uM)        0.2ul        
  Syb_FP5 (100uM)        0.2ul        
  H2O                     24ul       
  50X SYBG I             0.2ul

98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.

File:20090812 PhiX quantification.png
20090812_Parkinson_sample_quantification
No sample sample concentration
Par_1 5028 12.2nM
Par_2 4735 10.2nM
Par_3 4789 9.3nM
Par_4 5171 6.6nM
Par_5 1818 6.0nM
Par_6 1947 7.8nM
Par_7 1741 6.9nM
Par_8 4977 9.6nM
Par_9 4879 5.5nM
Par_10 4526 6.6nM