Kun:LabNotes/ExonomeSeq/2009-5-23: Difference between revisions

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===PCR===  
===PCR===  
                                   x2
                                   x2
  DNA                    10ul
  Solexa_PCR_up(10uM)      2ul
  Solexa_PCR_lo(10uM)      2ul
  2x iProof master mix    50ul
  50x SYBGI              0.4ul
  ddH2O                  36ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec)
  5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
==Added another round in parallel==
===PCR with dUTP===
                                      x 2 x 4
    Template:                  1.5ul
    2x EconoTaq Master Mix    50ul      400
    100uM AmpF6.3NH2          0.2ul      1.6
    100uM AmpR6.3NH2          0.2ul      1.6
    10mM dUTP                0.6ul      4.8
    50x SYBG I                0.4ul      3.2
    H2O                        48ul      350
    94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
    Purified every 200ul for each amplicon with one Qiaquick column.
 
    A: 54ng/ul x 28ul
    B: 45ng/ul x 28ul
    C: 40ng/ul x 28ul
    D: 38ng/ul x 28ul
===USER digestion===
    Qiaquick purified DNA        18ul
    USER enzyme                    2ul
    37C 1h
===S1 nuclease digestion===                                   
  10 x S1 nuclease buffer:  3ul   
  DNA after USER digestion: 20ul   
  S1 nuclease (10U/ul):      2ul   
  ddH2O                      4ul   
  37C 10mins.
  Purified with MinElute cloumn. Elute in 18ul H2O.
  A: 12ng/ul
  B: 7.4ng/ul
  C: 7.5ng/ul
  D: 12ng/ul
===End repair (Enzymatics End-repair Mix)===
  Fragmented DNA  15ul
  dNTP          2.5ul
  10x buffer    2.5ul
  Enzyme mix      1ul
  H2O              4ul
  Incubate at RT for 30 minutes
  Purified with Qiaquick column.
  Select the ~100bp fragments with 2% SizeSelect gel
===Adaptor ligation(Enzymatic T4 ligase)===
                            NEB   
  DNA                      34ul   
  2X Rapid Ligation buffer  40ul
  50uM Solexa_1 adaptor      2ul
  50uM Solexa_2 adaptor      2ul 
  T4 DNA ligase(600U/ul)    2ul
Keep at room temperature (~25C) for 15 minutes. Purify with MinElute columns.
===Nick translation===
  DNA                  8ul
  10x ThermoPol buffer 1ul
  10mM dNTP          0.4ul
  Bst Pol (8U/ul)    0.5ul 
  65C 10min
===PCR===                                   
   DNA                    10ul
   DNA                    10ul
   Solexa_PCR_up(10uM)      2ul
   Solexa_PCR_up(10uM)      2ul

Revision as of 22:40, 23 May 2009

Shotgun library construction

  • Start with four normalized and size-selected libraries captured from PGP1-3, NA12878 (prepared by Alice Li). The estimated concentration is ~2ng/ul each.

PCR with dUTP

                                      x 4 x 4
   Template:                   1ul
   2x EconoTaq Master Mix     50ul      800
   100uM AmpF6.3NH2          0.2ul      3.2
   100uM AmpR6.3NH2          0.2ul      3.2
   10mM dUTP                 0.6ul      9.6
   50x SYBG I                0.4ul      6.4
   H2O                        48ul      770
   94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified every 4x50ul for each amplicon with two MinElute column.
 
   A: 23ng/ul x 20ul
   B: 14ng/ul x 20ul
   C: 14ng/ul x 20ul
   D: 18ng/ul x 20ul
   Purified each of the remaining amplicons with one DNA concentrator-5 column, eluted with 20ul EB
   A: 6.3ng/ul x 20ul
   B: 6.2ng/ul x 20ul
   C: 6.8ng/ul x 20ul
   D: 4.8ng/ul x 20ul

USER digestion

   Qiaquick purified DNA         20ul
   USER enzyme                    2ul
   37C 1h

   DNA concentrator purified DNA         18ul
   USER enzyme                            1ul
   37C 1h

S1 nuclease digestion

 10 x S1 nuclease buffer:   3ul     
 DNA after USER digestion: 22ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      2ul     
 37C 15mins. Combined two tubes for each library.
 Purified with MinElute cloumn. Elute in 18ul H2O.
 A: 12ng/ul 
 B: 7.4ng/ul
 C: 7.5ng/ul
 D: 12ng/ul

End repair (Enzymatics End-repair Mix)

  Fragmented DNA  15ul
  dNTP           2.5ul
  10x buffer     2.5ul
  Enzyme mix       1ul
  H2O              4ul
  Incubate at RT for 30 minutes 
  Purified with Qiaquick column.
  Select the ~100bp fragments with 2% SizeSelect gel

Adaptor ligation(Enzymatic T4 ligase)

                            NEB     
  DNA                       20ul    
  2X Rapid Ligation buffer  25ul
  100uM Solexa_1 adaptor   0.5ul 
  100uM Solexa_2 adaptor   0.5ul   
  T4 DNA ligase(600U/ul)     1ul
  H2O                        3ul

Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.

Nick translation

  DNA                  8ul
  10x ThermoPol buffer 1ul
  10mM dNTP          0.4ul
  Bst Pol (8U/ul)    0.5ul  
  65C 10min

PCR

                                  x2
  DNA                     10ul
  Solexa_PCR_up(10uM)      2ul
  Solexa_PCR_lo(10uM)      2ul
  2x iProof master mix    50ul
  50x SYBGI              0.4ul
  ddH2O                   36ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 
  5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.

Added another round in parallel

PCR with dUTP

                                      x 2 x 4
   Template:                  1.5ul
   2x EconoTaq Master Mix     50ul      400
   100uM AmpF6.3NH2          0.2ul      1.6
   100uM AmpR6.3NH2          0.2ul      1.6
   10mM dUTP                 0.6ul      4.8
   50x SYBG I                0.4ul      3.2
   H2O                        48ul      350
   94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified every 200ul for each amplicon with one Qiaquick column.
 
   A: 54ng/ul x 28ul
   B: 45ng/ul x 28ul
   C: 40ng/ul x 28ul
   D: 38ng/ul x 28ul

USER digestion

   Qiaquick purified DNA         18ul
   USER enzyme                    2ul
   37C 1h

S1 nuclease digestion

 10 x S1 nuclease buffer:   3ul     
 DNA after USER digestion: 20ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      4ul     
 37C 10mins. 
 Purified with MinElute cloumn. Elute in 18ul H2O.
 A: 12ng/ul 
 B: 7.4ng/ul
 C: 7.5ng/ul
 D: 12ng/ul

End repair (Enzymatics End-repair Mix)

  Fragmented DNA  15ul
  dNTP           2.5ul
  10x buffer     2.5ul
  Enzyme mix       1ul
  H2O              4ul
  Incubate at RT for 30 minutes 
  Purified with Qiaquick column.
  Select the ~100bp fragments with 2% SizeSelect gel

Adaptor ligation(Enzymatic T4 ligase)

                            NEB     
  DNA                       34ul    
  2X Rapid Ligation buffer  40ul
  50uM Solexa_1 adaptor      2ul 
  50uM Solexa_2 adaptor      2ul   
  T4 DNA ligase(600U/ul)     2ul

Keep at room temperature (~25C) for 15 minutes. Purify with MinElute columns.

Nick translation

  DNA                  8ul
  10x ThermoPol buffer 1ul
  10mM dNTP          0.4ul
  Bst Pol (8U/ul)    0.5ul  
  65C 10min

PCR

  DNA                     10ul
  Solexa_PCR_up(10uM)      2ul
  Solexa_PCR_lo(10uM)      2ul
  2x iProof master mix    50ul
  50x SYBGI              0.4ul
  ddH2O                   36ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 
  5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.