Alice:LabNotes/2009-6-4

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Shotgun library construction

PCR with dUTP

                                          x 4     x4
   Template:                0.25ul        1ul
   2x EconoTaq Master Mix     50ul      200ul
   100uM AmpF6.3NH2          0.2ul      0.8ul
   100uM AmpR6.3NH2          0.2ul      0.8ul
   1mM dUTP                    4ul       16ul
   50x SYBG I                0.4ul      1.6ul
   H2O                        50ul      770ul
   94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified the amplicon with one Qiaquick column each
 
   e-1: 14.2ng/ul x 30ul
   e-2: 31.1ng/ul x 30ul
   f-1: 13.3ng/ul x 30ul
   f-2: 33.6ng/ul x 30ul

USER digestion

   Qiaquick purified DNA         15ul
   USER enzyme                    2ul
   37C 1h

S1 nuclease digestion

 10 x S1 nuclease buffer:   2ul  
 DNA after USER digestion: 17ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      2ul     
 37C 15mins
 Purified with MinElute cloumn. Elute in 18ul H2O.

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
 

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% Size Select E-gel. The total amount obtained is 25ul.

Nick translation

  DNA                  15ul
  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.


                                  x4
  DNA                      10ul (second half of the products)
  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) 
  5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.

Purify with one qiaquick column for each set, and elute in 40ul EB

a: 142.9ng/ul
b: 101.2ng/ul
c: 117.2ng/ul
d: 126.5ng/ul
perform gel size selection at 150-160bp
Ethanol precipitation on the sets from above, and also four tubes obtained from Dr. Zhang. Elute in 40ul of ddH2O each.