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

From ZhangLabWiki
Jump to navigation Jump to search
 
(9 intermediate revisions by the same user not shown)
Line 40: Line 40:
   ddH2O                      2ul     
   ddH2O                      2ul     


   37C 15mins.
   37C 15mins. Combined two tubes for each library.
   Purified with MinElute cloumn. Elute in 18ul H2O.
   Purified with MinElute cloumn. Elute in 18ul H2O.


Line 57: Line 57:
   Purified with Qiaquick column.
   Purified with Qiaquick column.
   Select the ~100bp fragments with 2% SizeSelect gel
   Select the ~100bp fragments with 2% SizeSelect gel
===Adaptor ligation(Enzymatic T4 ligase vs. NEB QuickLigase)===
===Adaptor ligation(Enzymatic T4 ligase)===
                             NEB     
                             NEB     
   DNA                      20ul    
   DNA                      34ul    
   2X Rapid Ligation buffer  25ul
   2X Rapid Ligation buffer  40ul
   100uM Solexa_1 adaptor   0.5ul
   50uM Solexa_1 adaptor     2ul
   100uM Solexa_2 adaptor   0.5ul    
   50uM Solexa_2 adaptor     2ul    
   T4 DNA ligase(600U/ul)    1ul
   T4 DNA ligase(600U/ul)    2ul
  H2O                        3ul
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.
===Nick translation===
===Nick translation===
   DNA                  8ul
   DNA                  8ul
Line 73: Line 73:
   65C 10min
   65C 10min
===PCR===  
===PCR===  
                                   x2
                                   x2 x 4
   DNA                     10ul
   DNA                     5ul
   Solexa_PCR_up(10uM)     2ul
   Solexa_PCR_up(100uM)   0.2ul
   Solexa_PCR_lo(10uM)     2ul
   Solexa_PCR_lo(100uM)   0.2ul
   2x iProof master mix    50ul
   2x iProof master mix    50ul
   50x SYBGI              0.4ul
   50x SYBGI              0.4ul
   ddH2O                  36ul
   ddH2O                  36ul
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec)  
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)  
   5 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==
 
===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: 11ng/ul
  B: 9ng/ul
  C: 7ng/ul
  D: 5ng/ul
 
  Hold the samples at this step.

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.