Alice:LabNotes/2009-5-24

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Revision as of 08:28, 25 May 2009 by >Zsakura2 (New page: ==Shotgun library construction== ===PCR with dUTP=== x 4 x 4 Template: 1ul 2x EconoTaq Master Mix 50ul 800 1...)
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Shotgun library construction

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                 0.4ul      6.4
   50x SYBG I                0.4ul      6.4
   H2O                        48ul      770
   94C 3min -> 11 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified the amplicon with one Qiaquick column each
 
   A: 82ng/ul x 30ul
   B: 51ng/ul x 30ul
   C: 59ng/ul x 30ul
   D: 85ng/ul x 30ul

USER digestion

   Qiaquick purified DNA         15ul
   USER enzyme                    2ul (3 ul used for tube a and d)
   37C 1h

S1 nuclease digestion

 10 x S1 nuclease buffer:   2ul    (2.3ul used for tube a and d)  
 DNA after USER digestion: 17ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      2ul     
 37C 15mins
 Purified with MinElute cloumn. Elute in 18ul H2O.
 a: 30ng/ul 
 b: 20ng/ul
 c: 24ng/ul
 d: 23ng/ul

End repair (epicentre)

  Fragmented DNA  15ul
  dNTP           2.5ul
  10x buffer     2.5ul
  enzyme         0.5ul
  ATP              5ul
  Incubate at RT for 40 minutes 
  Purified with minelute column and elute in 20ul
  Select the ~100bp fragments with TBE gel
 File:ZhangLab 2 2009-05-24 17hr 57min.jpg

Adaptor ligation

  DNA                        20ul    
  2X Rapid Ligation buffer   27ul
  100uM Solexa_1 adaptor      1ul 
  100uM Solexa_2 adaptor      1ul   
  QuickLigase(400U/ul)        2ul
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB.
performed one round of size selection on positive control tube using 2%SizeSelect E-gel. The total amount obtained is 25ul.

Nick translation

  DNA                  8ul
  10x ThermoPol buffer 2ul (3ul for positive control)
  10mM dNTP          0.4ul
  BSA                  2ul
  Bst Pol (8U/ul)      1ul  
  65C 20min

PCR

                             x 5
  DNA                      10ul
  Solexa_PCR_up(100uM)     0.2ul
  Solexa_PCR_lo(100uM)     0.2ul
  2x iProof master mix      50ul
  50x SYBGI                0.4ul
  ddH2O                     40ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 
  6 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
File:ZhangLab 2 2009-05-25 01hr 10min.jpg