Alice:LabNotes/2009-6-4

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single-end Shotgun sequencing library construction[edit]

PCR with dUTP[edit]

                                          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      200ul
   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
File:ZhangLab 2 2009-06-05 11hr 19min.jpg

USER digestion[edit]

   Qiaquick purified DNA         30ul
   USER enzyme                    2ul
   37C 1h

S1 nuclease digestion[edit]

 10 x S1 nuclease buffer:   4ul  
 DNA after USER digestion: 32ul    
 S1 nuclease (10U/ul):      1ul     
 ddH2O                      3ul     
 37C 15mins
 Purified with MinElute column. Elute in 18ul H2O.
Nanodrop: 
e-1:5ng/ul     e-2:7.1ng/ul
f-1:6.8ng/ul   f-2:10.1ng/ul

End repair (epicentre)[edit]

                                       pos. control
  Fragmented DNA  15ul             Spacer 107bp  2ul + H2O 13ul
  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 Egel (only the seq. library samples, positive control not included).

Adaptor ligation[edit]

                                      pos. control  neg control    
  DNA                        16ul         20ul        H2O 16ul       
  2X Rapid Ligation buffer   18ul         22ul           18ul
  100uM Solexa_1 adaptor     0.5ul       0.5ul           0.5ul
  100uM Solexa_2 adaptor     0.5ul       0.5ul           0.5ul
  QuickLigase(400U/ul)       1ul          1ul            1ul
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 16ul.

Nick translation[edit]

  DNA                  15ul
  10x ThermoPol buffer 2ul
  10mM dNTP          0.4ul
  BSA                  2ul
  Bst Pol (8U/ul)      1ul  
  65C 20min
TBE gel size selection at 150-170bp

PCR[edit]

                                 x 6
  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 -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 7 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold.
File:ZhangLab 2 2009-06-05 16hr 02min.jpg
                                     x 6
  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 -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 6 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold.
File:ZhangLab 2 2009-06-07 19hr 24min.jpg
Qiaquick column purification and elute in 40ul of EB
Nanodrop:
e-1: 24.8ng/ul 
e-2: 34.4ng/ul
f-1: 30.0ng/ul
f-2: 34.7ng/ul

result[edit]

gel quantification result:
e-1: PGP5-1-060809 (7.43ng/ul = 62.5nM)
e-2: PGP5-2-060809 (7.81ng/ul = 65.7nM)
f-1: PGP7-1-060809 (8.98ng/ul = 75.5nM)
f-2: PGP7-2-060809 (9.47ng/ul = 79.7nM)
send out 10ul of each for sequencing (for lane 4-7)
and also 10ul of f-2: PGP7-2-060809 for sequencing (for lane 8)