Alice:LabNotes/2009-5-24

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Shotgun library construction[edit]

Single-end sequencing libraries

PCR with dUTP[edit]

                                      x 4 x 4
   Template:                   1ul
   2x EconoTaq Master Mix     50ul      800
   100uM AmpF6.3NH2          0.2ul      3.2
   100uM AmpR6.3NH2          0.2ul      3.2
   10mM dUTP                 0.4ul      6.4
   50x SYBG I                0.4ul      6.4
   H2O                        48ul      770
   94C 3min -> 11 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified the amplicon with one Qiaquick column each
 
   A: 82ng/ul x 30ul
   B: 51ng/ul x 30ul
   C: 59ng/ul x 30ul
   D: 85ng/ul x 30ul

USER digestion[edit]

   Qiaquick purified DNA         15ul
   USER enzyme                    2ul (3 ul used for tube a and d)
   37C 1h

S1 nuclease digestion[edit]

 10 x S1 nuclease buffer:   2ul    (2.3ul used for tube a and d)  
 DNA after USER digestion: 17ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      2ul     
 37C 15mins
 Purified with MinElute cloumn. Elute in 18ul H2O.
 a: 30ng/ul 
 b: 20ng/ul
 c: 24ng/ul
 d: 23ng/ul

End repair (epicentre)[edit]

  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
 File:ZhangLab 2 2009-05-24 17hr 57min.jpg

Adaptor ligation[edit]

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

Nick translation[edit]

  DNA                  15ul
  10x ThermoPol buffer 2ul (3ul for positive control)
  10mM dNTP          0.4ul
  BSA                  2ul
  Bst Pol (8U/ul)      1ul  
  65C 20min

PCR[edit]

                                 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.
File:ZhangLab 2 2009-05-25 01hr 10min.jpg 
                                  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.

result[edit]

gel quantification:
1: 2ul low mass ladder + 2ul H2O + 2ul loading dye
2: 1ul low mass ladder + 2ul H2O + 2ul loading dye
3-6: 1ul DNA + 3ul H2O + 2ul loading dye
7-10: 1ul DNA + 3ul H2O + 2ul loading dye
 File:ZhangLab 2 2009-05-25 12hr 29min.jpg
yield:
a-k: 1.13 ng/ul = 10.71nM
b-k: 1.45 ng/ul = 13.73nM
c-k: 0.77 ng/ul = 7.27nM
d-k: 5.2 ng/ul = 49.25nM
a-A: 16.38 ng/ul = 155.1nM
b-A: 14.09 ng/ul = 133.41nM
c-A: 8.14 ng/ul = 77.1nM
d-A: 12.25 ng/ul = 116.04nM
A total of 8 tubes of solexa sequencing libraries were sent to BioGem.
(calculated by Dr. Zhang)
[tube 1]: PGP1_062509k,    19nM, 40ul
[tube 2]: PGP2_062509k,    20nM, 40ul
[tube 3]: PGP3_062509k,    19nM, 40ul
[tube 4]: NA12878_062509k, 33nM, 40ul
[tube 5]: PGP1_062509a,    77nM, 40ul
[tube 6]: PGP2_062509a,    64nM, 40ul
[tube 7]: PGP3_062509a,    49.5nM, 40ul
[tube 8]: NA12878_062509a, 60nM, 40ul