Alice:LabNotes/2009-6-4

From ZhangLabWiki
Revision as of 17:23, 5 June 2009 by >Sam Chiang (→‎Adaptor ligation)
Jump to navigation Jump to search

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         30ul
   USER enzyme                    2ul
   37C 1h

S1 nuclease digestion

 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)

  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 Egel.

Adaptor ligation

                                      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

  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.
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.