Dinh/NOTES/2010-8-19

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N2 Adaptor Protocol for Library Contruction on 220K captured PGP1-ips and PGP1-fib libraries[edit]

Experiment Plan:[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. 
5. 3.22ng/ulx20ul=64ng   (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected)
6. 1.18ng/ulx20ul=24ng   (PGP1-fib captured at 58C, 44ng 220K probes (30:1), size selected)
------------------------
Will not use 5 & 6
7. 10.89ng/ulx20ul=327ng (PGP1-fib captured at 58C, 176ng 220K probes (110:1), size selected)
8. 12.53ng/ulx30ul=376ng (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss)
9. 4.28ng/ulx30ul=129ng  (PGP1-fib captured at 60C, 44ng 220K probes (30:1), non-ss)
------------------------
Use 7 (B) and (C) combine 8 & 9 to get 505 ng in 60ul

Mme I digestion[edit]

1 units per 1ug phiX 174 DNA
1 phiX = 5 sites/5389bp
1 amplicon = 2 sites/300bp

7.2 units MmeI to digest 1ug capure PCR amplicons => 2.16 units to digest 300ng.
(A) 27ul capture amplicon + 4.8ul H2O (300ng)
(B) 17ul capture amplicon + 14.8ul H2O (185ng)
(C) 31.8ul capture amplicon (268ng)
  VOL     REAGENT      Conc            Final
31.8 ul DNA + H2O                      ~300ng
 4.0 ul NEBuffer 4      10x             1x 
 1.2 ul MmeI         2 units/ul        2.4 units
 3.0 ul SAM             1mM            75uM
 -------------------------------------------- 
40.0 ul TOTAL
Incubate at 37C for 1 hr. Purify with 1 MinElute column each.
Yield: (exp. 70%) A. 92ng (31%) | B. 52ng (28%) | C. 118ng (44%)

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

Est length of ligated products: 250bp (after MmeI) + 2x33bp = 316bp
MW ligased products = (316bp*607.4 D/bp +157.9 D) = 19.2kD = 19,200 g/mole
For 100 ng ligased product = 100ng / 19,200g/mole =  5.21E-3 nmole * 20:1 = 0.1042 nmole adapters required.
Adapters (ul) = 0.1042 / (20xE3nmoles/L * 1E-6L/ul) = 0.1042nmoles/ (20E-3 nmoles/ul) = 5.2 ul 
-----> 5.2 ul of 20uM adapters per 100ng ligased products. 

Ligation Reaction

   DNA           Adapters          QuickLigase          2xQuickLigase Buffer
A. 16ul          3.9ul             2ul                  21.9ul
B. 16ul          2.2ul             1ul                  19.2ul
C. 16ul          4.9ul             2ul                  22.9ul 
Incubate at RT for 15 min. Purify with 1 MinElute column each, elute with 15ul EB.

Yield (inaccurate because high amount of adapters added). 

Amplification[edit]

  1x   4x   Reagents
 1.0ul ---  adapter ligased DNA
 2.0ul 8    PCR_F(10uM) 
 2.0ul 8    PCR_R(10uM) 
 0.4ul 1.6  SYBR Green 50x
44.6ul 179  H2O
50.0ul 200  Phusion HF, 2xMM
--------------------------------
100 ul each
98C - 30s
98C - 10s
62C - 20s
72C - 17s
measure pt
72C - 3s
Go to 2, 10x
72C - 2 min
15C forever
Amplify for 8 cycles. Purify with 1 QiaQuick column.

File:DD 2010-08-20 N2 adaptor protocol.jpg