Alice:LabNotes/2009-10-9

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iPS library construction[edit]

The five libraries were pooled together as the following ratio (second PCR-amplification was not performed,
these are amplified capture product)
pooled all the subsets together using the ratio: 
1-100%(Jan09 1+2+3); 2-50%(Jan09 4+5DS); 3-73%(Jan09 6+7); 4-73%(Mar09 2+3, Jan09 5SS)
pooled all the subsets together using the ratio: 1-100%; 2-50%; 3-73%; 4-72%

                        DF1          DF2          DF3           DF4         
concnetration(ng/ul): 32.9ng/ul      14.7ng/ul    41.4ng/ul     12.9ng/ul
volume needed (ul):   4ul            4.5ul        2.3ul         7.5ul
--------------------------------------------------------------------------
total (ng):           132ng          66ng         96ng          96ng

                        FS1          FS2          DF3          DF4         
concnetration(ng/ul): 28ng/ul        13.8ng/ul    26.1ng/ul    9.4ng/ul
volume needed (ul):   7ul            7ul          5.4ul        5.6ul
--------------------------------------------------------------------------
total (ng):           86.1ng         43ng         63ng         63ng

                       ips 1         ips 2         ips 3       ips 4      
concnetration(ng/ul):  21.7ng/ul     9.7ng/ul     20.2ng/ul    7.9ng/ul
volume needed (ul):    6ul           6.5ul        4.5ul        10ul
--------------------------------------------------------------------------
total (ng):            130.2ng       65.1ng       95ng         95ng

                       CV-ips 1      CV-ips 2     CV-ips 3     CV-ips 4        
concnetration(ng/ul):  31.3ng/ul     10.2ng/ul    25ng/ul      12ng/ul
volume needed (ul):    4ul           6.1ul        3.7ul        7ul
--------------------------------------------------------------------------
total (ng):            125.2ng       62.6ng       91.4ng       91.4ng

                       CV-fb 1        CV-fb 2      CV-fb 3      CV-fb 4       
concnetration(ng/ul):  28ng/ul        13.8ng/ul    26.1ng/ul    9.4ng/ul
volume needed (ul):    4ul            4ul          3.1ul        8ul
--------------------------------------------------------------------------
total (ng):            112ng          56ng         82ng         81ng
Qiaquick column purification

MmeI digestion[edit]

DNA                  23ul     
10x NEB buffer 4     4ul      
1mM SAM              6ul     
2U/ul MmeI           8ul     
MinElute purification and elute in 100ul EB.
  • Sent out the five libraries to Harvard for Covaris shearing
  • Received five sheared samples from Billy, ~400ng in ~80ul each
  • Use the NEBNext DNA Sample Prep Master Mix Set I kit for library construction.
  • Use the Y-adaptor for ligation.

End repair[edit]

  DNA                    ~50ul
  End Repair 10X buffer   10ul
  End Repair Enzyme Mix    5ul
  H2O                    ~30ul
  Total                  100ul
  Room temperature for 20min, purified with Qiaquick column, eluted with 39ul EB

A Tailing[edit]

  End repaired DNA         37ul
  dA-Tailing buffer (10x)   5ul
  Klenow exo-               3ul
  H2O                       5ul
  Incubate at 37C for 30min
  • Purify with Qiaquick columns, eluted with 30ul EB.
  • Perform size selection on 20ul DNA with 2% size selection gel, extract the ~100bp bands.

Ligation[edit]

  • Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors.
  • Include a positive control.
  A-tailed DNA           15ul
  5X QuickLigase buffer  10ul
  20uM Adaptor mix        5ul
  Quick T4 ligase         5ul
  H2O                    15ul
  RT for 15min
  Purified with MinElute columns, eluted with 15ul EB.

PCR[edit]

Check the size distribution of the PCR products with 1ul of ligation products first
    Template                  1ul      
    2x iProof mix            50ul       
    Solexa_PCR_up (10uM)    0.4ul        
    Solexa_PCR_lo_PE (10uM) 0.4ul        
    H2O                      49ul     
    50X SYBG I              0.4ul     
    98C 30sec -> 13 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec)
File:ZhangLab 2 2009-10-09 15hr 40min.jpg
Repeat the PCR with real samples
    Template                  5ul      
    2x iProof mix            50ul       
    Solexa_PCR_up (10uM)    0.4ul        
    Solexa_PCR_lo_PE (10uM) 0.4ul        
    H2O                      45ul     
    50X SYBG I              0.4ul     
    98C 30sec -> 11 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec)
gel size selection: 
File:ZhangLab 2 2009-10-11 15hr 37min.jpg File:ZhangLab 2 2009-10-11 15hr 38min.jpg
File:ZhangLab 2 2009-10-11 16hr 05min.jpg

QPCR quantification[edit]

    Template                  1ul      
    2x iProof mix            25ul       
    Solexa_PCR_up (10uM)    0.2ul        
    Solexa_PCR_lo_PE (10uM) 0.2ul        
    H2O                      24ul     
    50X SYBG I              0.2ul     
    98C 30sec -> 20 cycles of (98C 10sec -> 62C 20sec -> 72C 15 sec) -> 72C 3min