Alice:LabNotes/2009-10-9: Difference between revisions

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==iPS library construction==
==iPS library construction==


  The five libraries were pooled together as the following ratio (second PCR-amplification was not performed,these are amplified capture product)
  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:  
  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)
  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%
  pooled all the subsets together using the ratio: 1-100%; 2-50%; 3-73%; 4-72%
 
                         DF1          DF2          DF3          DF4         
                         DF1          DF2          DF3          DF4         
  concnetration(ng/ul): 32.9ng/ul      14.7ng/ul    41.4ng/ul    12.9ng/ul
  concnetration(ng/ul): 32.9ng/ul      14.7ng/ul    41.4ng/ul    12.9ng/ul
Line 14: Line 14:
  total (ng):          132ng          66ng        96ng          96ng
  total (ng):          132ng          66ng        96ng          96ng
   
   
                         FS1          FS2          DF3          DF4         
                         FS1          FS2          DF3          DF4         
  concnetration(ng/ul): 28ng/ul        13.8ng/ul    26.1ng/ul    9.4ng/ul
  concnetration(ng/ul): 28ng/ul        13.8ng/ul    26.1ng/ul    9.4ng/ul
Line 20: Line 19:
  --------------------------------------------------------------------------
  --------------------------------------------------------------------------
  total (ng):          86.1ng        43ng        63ng        63ng
  total (ng):          86.1ng        43ng        63ng        63ng
 
                         ips 1        ips 2        ips 3      ips 4       
                         ips 1        ips 2        ips 3      ips 4       
  concnetration(ng/ul):  21.7ng/ul    9.7ng/ul    20.2ng/ul    7.9ng/ul
  concnetration(ng/ul):  21.7ng/ul    9.7ng/ul    20.2ng/ul    7.9ng/ul
Line 26: Line 25:
  --------------------------------------------------------------------------
  --------------------------------------------------------------------------
  total (ng):            130.2ng      65.1ng      95ng        95ng
  total (ng):            130.2ng      65.1ng      95ng        95ng
 
                         CV-ips 1      CV-ips 2    CV-ips 3    CV-ips 4         
                         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
  concnetration(ng/ul):  31.3ng/ul    10.2ng/ul    25ng/ul      12ng/ul
Line 32: Line 31:
  --------------------------------------------------------------------------
  --------------------------------------------------------------------------
  total (ng):            125.2ng      62.6ng      91.4ng      91.4ng
  total (ng):            125.2ng      62.6ng      91.4ng      91.4ng
 
                         CV-fb 1        CV-fb 2      CV-fb 3      CV-fb 4       
                         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
  concnetration(ng/ul):  28ng/ul        13.8ng/ul    26.1ng/ul    9.4ng/ul
Line 73: Line 72:


===Ligation===
===Ligation===
*Mix 20ul A_adaptor_b (100uM), 20ul A_adaptor_t (100uM), 5ul 10x Taq buffer, 55ul H2O.
 
*94C 3min -> ramp to 20C at 0.1C/sec -> 4C hold.
*Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors.
*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.
*Include a positive control.
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===PCR===
===PCR===
#Check the size distribution of the PCR products with 1ul of ligation products first
Check the size distribution of the PCR products with 1ul of ligation products first
     Template                  1ul       
     Template                  1ul       
     2x iProof mix            50ul       
     2x iProof mix            50ul       
Line 95: Line 93:
     98C 30sec -> 13 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec)
     98C 30sec -> 13 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec)
  [[Image:ZhangLab_2 2009-10-09 15hr 40min.jpg]]
  [[Image: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:
[[Image:ZhangLab_2 2009-10-11 15hr 37min.jpg]] [[Image:ZhangLab_2 2009-10-11 15hr 38min.jpg]]
[[Image:ZhangLab_2 2009-10-11 16hr 05min.jpg]]
===QPCR quantification===
    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

Latest revision as of 00:55, 20 October 2009

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