Alice:LabNotes/2009-6-10

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single-end Shotgun sequencing library construction[edit]

  • capture experiment and template information can be found under LabNote 6/4/09

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 -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified the amplicon with one Qiaquick column each
 
   e-1: 75.2ng/ul x 30ul
   e-2: 52.6ng/ul x 30ul
   f-1: 49.6ng/ul x 30ul
   f-2: 62.8ng/ul x 30ul
File:ZhangLab 2 2009-06-09 12hr 01min-dutp.jpg

USER digestion[edit]

   Qiaquick purified DNA         30ul
   USER enzyme                    3ul
   37C 1h

S1 nuclease digestion[edit]

 10 x S1 nuclease buffer:   4ul  
 DNA after USER digestion: 33ul    
 S1 nuclease (10U/ul):      1ul     
 ddH2O                      2ul     
 37C 15mins
 Purified with MinElute column. Elute in 18ul H2O.

End repair (epicentre)[edit]

                                       pos. control
  Fragmented DNA  18ul             Spacer 107bp 3ul + H2O 12ul
  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      
  DNA                        16ul         20ul              
  2X Rapid Ligation buffer   18ul         22ul          
  100uM Solexa_1 adaptor     0.5ul       0.5ul          
  100uM Solexa_2 adaptor     0.5ul       0.5ul         
  QuickLigase(400U/ul)       1ul          1ul          
  ATP                        3ul          3ul
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 of samples (not including control) at ~160bp

PCR[edit]

                                  x5
  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 2009-06-10 12hr 31min.jpg

qiauqick column purification combining the two PCR tubes for one sample, and elute in 30ul of EB.
nanodrop:
a3: 17.3ng/ul
b3: 18.2ng/ul
c3: 18.4ng/ul
d3: 22ng/ul
gel quantification and final concentration estimate (sent to Zhen Ye):
lane 1:  PGP1-3-061009: 34nM x 15ul;   (4ng/ul)
lane 2:  PGP2-3-061009: 25nM x 15ul;   (3ng/ul)
lane 3:  PGP3-3-061009: 22nM x 15ul;   (2.6ng/ul)
lane 4:  NA12878-3-061009: 22nM x 15ul;   (2.6ng/ul)