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

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Line 82: Line 82:
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)  
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)  
   9 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
   9 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
  Purified 200ul amplicons with one Qiaquick column.
  Yield:
  A: 69ng/ul x 40ul
  B: 81ng/ul x 40ul
  C: 83ng/ul x 40ul
  D: 58ng/ul x 40ul
  Perform size selection on half of the ampilcons with SizeSelect gel.
  Quantify the selected fragments with 6% TBE gel, use the amplicon size of 160bp.
  Divided the concentration by 2 for the tube sent to BioGem.
  A: 9.7ng/ul  => 92nM    /2 => 46nM
  B: 6.2ng/ul  => 58nM    /2 => 29nM
  C: 8.5ng/ul  => 81nM    /2 => 40nM
  D: 9.1ng/ul  => 86nM    /2 => 82nM
  Perform size selection on the second half of the ampilcons with PAGE gel.


==Added another round in parallel==
==Added another round in parallel==
Line 122: Line 139:


   Hold the samples at this step.
   Hold the samples at this step.
===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.

Latest revision as of 01:32, 24 May 2009

Shotgun library construction[edit]

  • 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[edit]

                                      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[edit]

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

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

S1 nuclease digestion[edit]

 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)[edit]

  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)[edit]

                            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 10 minutes. Purify with MinElute columns.

Nick translation[edit]

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

PCR[edit]

                                  x2 x 4
  DNA                      5ul
  Solexa_PCR_up(100uM)   0.2ul
  Solexa_PCR_lo(100uM)   0.2ul
  2x iProof master mix    50ul
  50x SYBGI              0.4ul
  ddH2O                   36ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 
  9 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
 Purified 200ul amplicons with one Qiaquick column.
 Yield:
 A: 69ng/ul x 40ul
 B: 81ng/ul x 40ul
 C: 83ng/ul x 40ul
 D: 58ng/ul x 40ul
 Perform size selection on half of the ampilcons with SizeSelect gel.
 Quantify the selected fragments with 6% TBE gel, use the amplicon size of 160bp.
 Divided the concentration by 2 for the tube sent to BioGem.
 A: 9.7ng/ul  => 92nM    /2 => 46nM
 B: 6.2ng/ul  => 58nM    /2 => 29nM
 C: 8.5ng/ul  => 81nM    /2 => 40nM
 D: 9.1ng/ul  => 86nM    /2 => 82nM
 Perform size selection on the second half of the ampilcons with PAGE gel.

Added another round in parallel[edit]

PCR with dUTP[edit]

                                      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[edit]

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

S1 nuclease digestion[edit]

 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: 11ng/ul 
 B: 9ng/ul
 C: 7ng/ul
 D: 5ng/ul
 Hold the samples at this step.