Alice:LabNotes/2009-6-4: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Zsakura2
>Sam Chiang
Line 1: Line 1:
==Shotgun library construction==
==single-end Shotgun sequencing library construction==




Line 70: Line 70:


  TBE gel size selection at 150-170bp
  TBE gel size selection at 150-170bp


===PCR===  
===PCR===  
                                   x 5
                                   x 6
   DNA                     10ul
   DNA                       10ul
   Solexa_PCR_up(100uM)    0.2ul
   Solexa_PCR_up(100uM)    0.2ul
   Solexa_PCR_lo(100uM)    0.2ul
   Solexa_PCR_lo(100uM)    0.2ul
Line 82: Line 83:


  [[Image:ZhangLab_2 2009-06-05 16hr 02min.jpg]]
  [[Image: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.
[[Image: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===
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: PGP7-2-060809 (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)

Revision as of 19:57, 8 June 2009

single-end Shotgun sequencing 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
File:ZhangLab 2 2009-06-05 11hr 19min.jpg

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)

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

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

PCR

                                 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

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: PGP7-2-060809 (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)