Alice:LabNotes/2009-7-21: Difference between revisions

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>Jie deng
(New page: ==Test the PCR-free library construction protocol== NA12878_070809: 100ul (7.08ng/ul) volume were added up to 100ul total with ddH2O (required by Covaris) Pos. CTRL: 107bp spacer (30ng/...)
 
>Zsakura2
 
(13 intermediate revisions by 3 users not shown)
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     Incubate at RT for 30-min
     Incubate at RT for 30-min
     Purified with QiqQuick columns, eluted with 37ul EB
     Purified with QiqQuick columns, eluted with 38ul EB
 
Nanodrop:
NA12878: 20.5ng/ul
positive control: 7ng/ul


===A-tail addition===
===A-tail addition===
Line 32: Line 36:
**Adaptor/insert ratio should be ~10:1, which would be roughly 2ul per 400ng sonicated DNA.
**Adaptor/insert ratio should be ~10:1, which would be roughly 2ul per 400ng sonicated DNA.
                                         PGP5      Pos. CTRL    Neg. CTRL
                                         PGP5      Pos. CTRL    Neg. CTRL
       DNA  (~170ng)                      11ul        11ul        11ul H2O   
       DNA  (~500ng)                      11ul        11ul        11ul H2O   
       2x QuickLigase buffer (NEB)     13.5ul      13.5ul      13.5ul
       2x QuickLigase buffer (NEB)       16ul        16ul        16ul
       20uM Adaptor oligo mix              2ul         2ul         2ul
       20uM Adaptor oligo mix              3ul         3ul         3ul
       T4 DNA QuickLigase (NEB)            1ul         1ul         1ul
       T4 DNA QuickLigase (NEB)            2ul         2ul         2ul
       extra ATP                          5ul          5ul          5ul
       extra ATP                          5ul          5ul          5ul
       Incubate at RT for 15 minutes.
       Incubate at RT for 15 minutes.
       Purified with MinElute columns, eluted with 20ul EB
       Purified with MinElute columns, eluted with 20ul EB


 
===PCR of sequencing library===
===PCR of sequencing library
                                    x3
                           
     Template                 5ul        
     Template               10ul        
     2x iProof mix          50ul       
     2x iProof mix          50ul       
     Solexa_PCR_up (10uM)    2ul       
     Solexa_PCR_up (10uM)    2ul       
     Solexa_PCR_lo_PE (10uM)  2ul         
     Solexa_PCR_lo_PE (10uM)  2ul         
     H2O                    36ul      
     H2O                    41ul      
     50X SYBG I            0.2ul     
     50X SYBG I            0.2ul     


   98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 70C 15 sec) -> 10 cycles of (98C 10sec -> 70C 15 sec)->  
   98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 11 cycles of (98C 10sec -> 72C 15 sec)->  
   70C 3min -> 15C hold.
   72C 3min -> 15C hold.
[[Image:ZhangLab_2 2009-07-21 21hr 02min.jpg]]
 
Qiaquick column purification, elute in 30ul EB
Nanodrop: 274ng/ul (260/280: 1.91)
 
Egel size selection at around 200-225bp
gel quantification: 2.5ng/ul (estimated as 10ng/ul)
Qubit: 27ng/ul
 
===sequencing run===
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Amount loaded'''
| align="center" style="background:#f0f0f0;"|'''Library conc.'''
|-
| Lane 1||phiX||3pmole||
|-
| Lane 2||phiX||4pmole||
|-
| Lane 3||NA12878||4pmole||10nM
|-
| Lane 4||Hues6ES-DSp||5pmole||38nM
|-
| Lane 5||HUES6ES-tRp||5pmole||28nM
|-
| Lane 6||Hybrid1-EB||5pmole||606nM
|-
| Lane 7||Hybrid2-ES||5pmole||395nM
|-
| Lane 8||Hybrid2-EB||5pmole||303nM
|-
|
|}

Latest revision as of 21:43, 28 July 2009

Test the PCR-free library construction protocol[edit]

NA12878_070809: 100ul (7.08ng/ul)
volume were added up to 100ul total with ddH2O (required by Covaris)
Pos. CTRL: 107bp spacer (30ng/ul)

End repair (Enzymatic End-repair Mix)[edit]

                      PGP5     Pos. CTRL
   Sonicated DNA     100ul     25ulDNA + 75ul of H2O
   dNTP                5ul
   10x buffer         12ul
   Enzyme mix          5ul

   Incubate at RT for 30-min
   Purified with QiqQuick columns, eluted with 38ul EB
Nanodrop:
NA12878: 20.5ng/ul
positive control: 7ng/ul

A-tail addition[edit]

   Blunt-ended DNA        35ul
   10X Klenow buffer       5ul
   1mM dATP               10ul
   Klenow fragment (exo-)  1ul
   37C 30min, purified with MinElute columns, eluted with 12ul EB. 

TA ligation[edit]

  • Prepare 20uM adaptor oligo duplex.
     100uM A_adaptor_t         20ul
     100uM A_adaptor_b         20ul
     10x T4 ligase buffer       5ul
     H2O                       55ul
     94C 3min -> cool down to 20C at the rate of 0.05C/sec. 
  • TA ligation:
    • Adaptor/insert ratio should be ~10:1, which would be roughly 2ul per 400ng sonicated DNA.
                                        PGP5       Pos. CTRL     Neg. CTRL
     DNA  (~500ng)                      11ul         11ul         11ul H2O  
     2x QuickLigase buffer (NEB)        16ul         16ul         16ul
     20uM Adaptor oligo mix              3ul          3ul          3ul
     T4 DNA QuickLigase (NEB)            2ul          2ul          2ul
     extra ATP                           5ul          5ul          5ul
     Incubate at RT for 15 minutes.
     Purified with MinElute columns, eluted with 20ul EB

PCR of sequencing library[edit]

                                   x3
   Template                 5ul       
   2x iProof mix           50ul       
   Solexa_PCR_up (10uM)     2ul      
   Solexa_PCR_lo_PE (10uM)  2ul        
   H2O                     41ul     
   50X SYBG I             0.2ul     
 98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 11 cycles of (98C 10sec -> 72C 15 sec)-> 
 72C 3min -> 15C hold.
File:ZhangLab 2 2009-07-21 21hr 02min.jpg
Qiaquick column purification, elute in 30ul EB
Nanodrop: 274ng/ul (260/280: 1.91)
Egel size selection at around 200-225bp
gel quantification: 2.5ng/ul (estimated as 10ng/ul)
Qubit: 27ng/ul

sequencing run[edit]

' Sample Amount loaded Library conc.
Lane 1 phiX 3pmole
Lane 2 phiX 4pmole
Lane 3 NA12878 4pmole 10nM
Lane 4 Hues6ES-DSp 5pmole 38nM
Lane 5 HUES6ES-tRp 5pmole 28nM
Lane 6 Hybrid1-EB 5pmole 606nM
Lane 7 Hybrid2-ES 5pmole 395nM
Lane 8 Hybrid2-EB 5pmole 303nM