Dinh/NOTES/2010-8-23

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MmeI Digestion[edit]

 File:PGP1-ips, PGP1-f 220k capture lib quant.jpg
1. 10.16ng/ulx20ul=203ng (PGP1-iPS captured at 58C, 44ng 220K probes (30:1), size selected)
3. 12.76ng/ulx20ul=255ng (PGP1-iPS captured at 60C, 44ng 220K probes (30:1), size selected)
------------------------
(A) Combine 1&3 to get 458ng in 40ul PGP1-iPS captured amplicons. 
*Reamplified (A) for 10 cycles with NH2 primers: 109.8ng/ul x 35ul
   VOL     REAGENT      Conc            (A)          (B)         (C)        (D)         (E)
31.8 ul DNA + H2O                       3ul           3ul         6ul       6ul         3ul
 4.0 ul NEBuffer 4      10x                                   
 --- ul MmeI         2 units/ul        1.2ul(1x)     2.4ul(2x)  2.4ul(1x)  3.6ul(1.5x)  3.6ul(2x)
 3.0 ul SAM             1mM             
 -------------------------------------------- 
40.0 ul TOTAL
Incubate at 37C for 1 hr. Purify with 1 MinElute column each.
Yield: (B) 14.4ng/ulx12.5ul=60%, (E) 16.0ng/ulx13.0ul=69%

Expected Lengths[edit]

1. Digested products, (remove linkers (25bp) + 18/20bp) = 300-43-45 = 212bp
2. Adapters ligased products: digested product(212bp)+36(top)+36(bottom) = 284bp
3. Amplified with solexa tail: digested product(212bp)+64(left)+60(right) = 336bp

Ligation[edit]

Adaptors preparation:

 20ul PE_N2_adaptor (100uM)
 20ul PE_b_A (100uM)
 10ul stoffel buffer (10x)
 50ul H2O
 ----------------------------
 100 ul TOTAL (20uM adaptors)

94C 2min -> 0.2C/sec to 20C -> 4C hold
Adapters to ligated product ratio: 20:1
Previously calculated 6.6ul per 100ng digested product (too much!)
Est length of digested products: 214bp (after MmeI)
MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole
For 100 ng digested product = 100ng / 128,927g/mole =  7.76E-4 nmole * 20:1 = 0.02 nmole adapters required.
Adapters (ul) = 0.02nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.02nmoles/ (20E-3 nmoles/ul) = 0.77 ul 
-----> 0.77 ul of 20uM adapters per 100ng digested products. 
Added 100ng from (B) and (E) but actual digested product ~50% or 50ng.

Ligation Reaction

   DNA           Adapters          QuickLigase          2xQuickLigase Buffer
B. 7.20ul(100ng)  3.3ul              2ul                  12.50ul
E. 6.25ul(100ng)  3.3ul              2ul                  11.55ul
Incubate at RT for 15 min. Purify with 1 MinElute column each, elute with 15ul EB.

Amplification[edit]

  1x    Reagents
10.0ul  adapter ligased DNA
 2.0ul  PCR_F(10uM) 
 2.0ul  PCR_R(10uM) 
 0.4ul  SYBR Green 50x
35.6ul  H2O
50.0ul  Phusion HF, 2xMM
--------------------------------
100 ul each
98C - 30s
98C - 10s
62C - 20s
70C - 30s
Go to 2, 7x
72C - 2 min
15C forever
Amplify for 8 cycles. Purify with 1 QiaQuick column.