Noi/NOTES/2013-2-7

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Revision as of 23:12, 13 February 2013 by >Noi
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Skin Microbiom Project

Library construction (72 samples)

  • After DNA shearing on 96-well microtubes, I did bead purification and resuspend DNA in 50ul.
    • There were some problem during transfer samples from micro-tubes to 96-well plate. The aluminum foil I used to seal the plate had a very bad quality, since it left the sticky part cover the well after I unsealed it. This block the pipette tip to transfer the liquid. I need to manually make the whole with clean pipette tip to clear the well before transfering sample with the robot. Another problem is the robot didn't completely transfer the liquid to the well (may be I need to adjust the dispensing speed). It have the droplet at the tip and might drop and get contaminate to other wells (I observed this about 3-4 samples)
  • I randomly pick 10 samples to run the gel check (I used little amount of beads (saw the bead left in the well very little) but comparable to the previous time, so I kind of worry about if I will lose most of the DNA). Next time I may use undiluted beads instead since it will be re-used in the following steps of library prep.

File:ZhangLab 2 2013-02-07 12hr 45min check shearedDNA aftersheared beadpure.jpg

  • From the gem image, I got the intensity of DNA in all 10 samples, and it's pretty close to the rehearsal.

End repair

Total reaction 70.00 ul
DNA 50.00 ul
End repair reaction mix 20.00 ul
  • 20C for 30min
KAPA End repair mix 1x rxn 75x rxn mix
10X End repai buffer 7.00 525.00
KAPA End repair enzyme 5.00 375.00
H2O 8.00 600.00
Total 20.00 1500.00
  • Aliquot to column #1: 187ul

A-tailing

  • A-tailing reaction mix: Total 30ul/reaction
  • 30C for 30min
A-tailing reaction mix 1x rxn 75x rxn mix
KAPA 10X A-tailing buffer 3.00 225.00
KAPA A-tailing enzyme 3.00 225.00
H2O 24.00 1800.00
Total 30.00 2250.00
  • Aliquot to column #2 (for 40rxn): 155ul, column #3 (for 32 rxn): 124ul

Adapter ligation

  • Adapter ligation mix (Total 50ul) : Ligation reaction mix 40ul/reaction & 10ul of adapters
  • 20C for 15min
Ligation reaction mix 1x rxn 75x rxn mix
KAPA 5X Ligation buffer 10.00 750.00
KAPA DNA ligase 3.00 225.00
H2O 27.00 2025.00
Total 40.00 3000.00
  • Aliquot to column #4 (for 40rxn): 207ul, column #5 (for 32 rxn): 165ul

Adapter preparation

Assume DNA amount ~100ng, size 400bp
MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243118 g/mole
For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required.
Adapters (ul) = 0.0082nmoles/ (20xE3 nmoles/L * 1E-6L/ul) = 0.0082nmoles/ (20E-3 nmoles/ul) =  0.411ul
  • I will prepare total volume of 20uM annealed adapters 40ul
Components 10ul 40ul
Stoffel buffer 1.00 4.00
PE_t_adapter 2.00 8.00
PE_b_adapter 2.00 8.00
H2O 5.00 20.00
  • 94C 2min -> 0.2C/sec to 20C -> 4C hold
  • Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> I will prepare diluted adapters for ~90rxn)
20uM annealed adapter 4.10 (for 10 rxn 36.90 (for ~90 rxn)
10x Stoffel buffer 10.00 90.00
H2O 85.90 773.10
Total 100.00 900.00
  • Aliquot to column #6 = 112ul

Amplification

  • TEST if amplification works (in small volume 25ul add 3ul of adapter ligated template)
Components 1x rxn 20.00
adapter ligated DNA 1.25 0.00
PCR_F(10uM) 0.50 10.00
PCR_R.N2IndX(10uM) 0.50 10.00
H2O 10.25 205.00
2X KAPA SYBR MM 12.50 250.00
Total volume 25.00 500.00
Program'
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x10, 72C - 2min, hold at 15C
  • PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification)

File:ZhangLab 2 2013-02-08 16hr 24min TEST-PCR A3-H3 A4-H4 2.jpg

Sequencing library concentration by Nanodrop

  • Note: The combine equl molecular ratio of sequencing libraries, I measured concentration of each library by Nanodrop. The corresponding concentration may not be very accurate as PQ (at specific 300-600bp range) and the concentration may be overestimated. However, this should be good enough. For some libraries which have very low concentration (<5.5ng/ul), I add maximum volume 35ul to the library pool to save half of them for DSN nomalization.
Sample ID ng/ul Yields in 70ul (ng) Volume for 200ng (ul) Total amount (ng) Approximated volume left (ul)
a3 10.51 735.70 19.03 200.00 50.97
b3 12.93 905.10 15.47 200.00 54.53
c3 7.86 550.20 25.45 200.00 44.55
d3 6.56 459.20 30.49 200.00 39.51
f3 12.22 855.40 16.37 200.00 53.63
f3 12.60 882.00 15.87 200.00 54.13
g3 5.48 383.60 35.00 191.80 35.00
h3 8.37 585.90 23.89 200.00 46.11
a4 10.34 723.80 19.34 200.00 50.66
b4 16.43 1150.10 12.17 200.00 57.83
c4 40.51 2835.70 4.94 200.00 65.06
d4 30.31 2121.70 6.60 200.00 63.40
e4 20.63 1444.10 9.69 200.00 60.31
f4 22.15 1550.50 9.03 200.00 60.97
g4 15.48 1083.60 12.92 200.00 57.08
h4 13.09 916.30 15.28 200.00 54.72
a5 13.60 952.00 14.71 200.00 55.29
b5 29.56 2069.20 6.77 200.00 63.23
c5 15.63 1094.10 12.80 200.00 57.20
d5 30.58 2140.60 6.54 200.00 63.46
e5 29.43 2060.10 6.80 200.00 63.20
f5 16.55 1158.50 12.08 200.00 57.92
g5 11.05 773.50 18.10 200.00 51.90
h5 12.59 881.30 15.89 200.00 54.11
a6 12.06 844.20 16.58 200.00 53.42
b6 18.89 1322.30 10.59 200.00 59.41
c6 12.57 879.90 15.91 200.00 54.09
d6 13.98 978.60 14.31 200.00 55.69
e6 18.46 1292.20 10.83 200.00 59.17
f6 18.98 1328.60 10.54 200.00 59.46
g6 12.28 859.60 16.29 200.00 53.71
h6 4.43 310.10 35.00 155.05 35.00
a7 9.23 646.10 21.67 200.00 48.33
b7 20.87 1460.90 9.58 200.00 60.42
c7 19.38 1356.60 10.32 200.00 59.68
d7 33.00 2310.00 6.06 200.00 63.94
e7 16.55 1158.50 12.08 200.00 57.92
f7 16.68 1167.60 11.99 200.00 58.01
g7 13.04 912.80 15.34 200.00 54.66
h7 9.28 649.60 21.55 200.00 48.45
a8 5.89 412.30 33.96 200.00 36.04
b8 9.28 649.60 21.55 200.00 48.45
c8 10.88 761.60 18.38 200.00 51.62
d8 10.91 763.70 18.33 200.00 51.67
e8 10.18 712.60 19.65 200.00 50.35
f8 10.30 721.00 19.42 200.00 50.58
g8 3.65 255.50 35.00 127.75 35.00
h8 4.04 282.80 35.00 141.40 35.00
a9 5.71 399.70 35.03 200.00 34.97
b9 7.70 539.00 25.97 200.00 44.03
c9 13.63 954.10 14.67 200.00 55.33
d9 15.94 1115.80 12.55 200.00 57.45
e9 10.19 713.30 19.63 200.00 50.37
f9 14.79 1035.30 13.52 200.00 56.48
g9 14.80 1036.00 13.51 200.00 56.49
h9 10.99 769.30 18.20 200.00 51.80
a10 4.78 334.60 35.00 167.30 35.00
b10 10.83 758.10 18.47 200.00 51.53
c10 7.88 551.60 25.38 200.00 44.62
d10 13.33 933.10 15.00 200.00 55.00
e10 8.53 597.10 23.45 200.00 46.55
f10 10.81 756.70 18.50 200.00 51.50
g10 10.17 711.90 19.67 200.00 50.33
h10 6.92 484.40 28.90 200.00 41.10
a11 6.90 483.00 28.99 200.00 41.01
b11 9.62 673.40 20.79 200.00 49.21
c11 8.67 606.90 23.07 200.00 46.93
d11 12.25 857.50 16.33 200.00 53.67
e11 11.38 796.60 17.57 200.00 52.43
f11 11.91 833.70 16.79 200.00 53.21
g11 12.44 870.80 16.08 200.00 53.92
h11 9.89 692.30 20.22 200.00 49.78
  • Performed PAGE size-selection (200ng each from 72 libraries, in 4 of 5-well gels)
File:ZhangLab 2 2013-02-12 12hr 21min PAGE-ss1.jpg File:ZhangLab 2 2013-02-12 12hr 24min PAGE-ss2.jpg
  • Resuspend ~70ul with H20
  • Verify the size in 6% TBE gel
File:ZhangLab 2 2013-02-12 15hr 59min.jpg
  • Quantify concemtration by Qubit dsDNA HS assay
    • Average conc. of = 19.73ng/ul
    • Conc. based on MW (size ~400bp) 243,228g/mol = 81.1nM
  • Library ID: NP-SkinMB_N2_Ind17-88_Feb07.2013