Calendar/2011-3-28

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Library Construction on sample 1-21 from Dr. Heller[edit]

Qubit Quantification[edit]

  • Dilute 25ul 200X dye with 4975ul buffer
Sample Concentration(ng/ml) Sample Concentration (ug/ml)
1 37.2 7.44
2 103 20.6
3 Out Of Range
4 27 5.4
5 47.8 9.56
6 12.7 2.55
7 Out Of Range
8 Out Of Range
9 Out Of Range
10 Out Of Range
11 Out Of Range
12 Out Of Range
13 Out Of Range
14 Out Of Range
15 Out Of Range
16 Out Of Range
17 Out Of Range
18 Out Of Range
19 Out Of Range
20 Out Of Range
21 Out Of Range
  • Samples are too dilute

Concentrate Samples using speed vacuum centrifuge concentrator[edit]

  • Dry sample with speedvac.
  • Add 5ul ddH2Oto dry pellet

Low input transposase-based library preparation (Shendure's Protocol) for the low amount samples[edit]

  • 1ul genomic DNA
  • 1ul 1:50 Nextera illumina compatible transposomes (1 μl Nextera enzyme, 24 μl TE, 25 μl 100% gly- cerol)
  • 1ul 5X NExtera HMW buffer
  • 2ul nuclease-free water
  • Incubate at 55C for 5 min


  • 12.5ul 2X Nextera PCR buffer
  • 0.5ul SYBR Green
  • 0.5ul 50X Nextera primer cocktail
  • 0.5ul 0.5uM barcode adaptor 2
  • 0.5μl Nextera PCR Enzyme (sold separately, see Related Products p. 8)

Cycle under standard Nextera conditions for 20 cycles Clean up using Minelut,elute in 20ul EB

Tagmentation Reaction for samples with high concentration[edit]

Sample Concentration(ng/ul) To get 50ng
1 7.44 6.72
2 20.6 2.43
4 5.4 9.26
5 9.56 5.23
6 2.55 19.61
h2o
8.28
12.57
5.74
9.77
-4.61
  • Prior to assembling the reaction, briefly centrifuge the 5X Nextera Reaction Buffer and Nextera Enzyme Mix tubes to assure that the reagents are at the bottom of the tubes.
  • Assemble the following reaction components on ice, in the order listed:
x µl Nuclease-Free Water 
50 ng Target DNA (in T10E1 Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA])
4 µl 5X Nextera Reaction Buffer LMW or HMW (see Important Considerations, no. 1, p. 3)
1 µl Nextera Enzyme Mix (Illumina-compatible)
20 µl Total reaction volume
  • Mix by vortexing, incubate at 55C for 5min
  • purify using minElute
  • Elute with 11ul EB

Add bPCR-compatible sites and optional bar coding by PCR[edit]

  • Assemble the following reaction components at room temperature:
17 μl Nuclease-Free water 
5 μl Recovered DNA Fragment Library (from Step A3)
25 μl 2X Nextera PCR Buffer 
1 μl 50X Nextera Primer Cocktail (Illumina-compatible) 
1 μl bar coded Illumina-compatible Adaptor 2
1 μl Nextera PCR Enzyme (sold separately, see Related Products p. 8)
50 μl Total reaction volume
Sample Barcode
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 1
12 2
13 3
14 4
15 5
16 6
17 7
18 8
19 9
20 10
21 11

PCR[edit]

72C 3min
95C 30sec
9X
95C 10 sec
62C 30 sec
72C 3 min
hold at 4C
  • purify tagged DNA with minelute, elute with ul EB

Results[edit]

  • libraries for high starting amount were amplified

File:040511 P1.jpg

  • libraries for low starting amount didn't amplify (Cycle1-20)

File:040511 P2.jpg

  • Added 5 more cycles

File:040511 P3.jpg

  • Page Gel quantification

File:ZhangLab 2 2011-04-05 18hr 05min-alan1 low.jpg File:ZhangLab 2 2011-04-05 18hr 03min-alan2.jpg

Purify Sample # 1,2,4,5&6 to remove primer dimers[edit]

Qubit Quantification[edit]

Sample Concentration in the Qubit µL used Dilution Sample Concentration (ng/ul)
1 51.6 ng/mL 1 200 10.3
2 34.6 ng/mL 1 200 6.92
3 0.58 ng/mL 1 200 0.12
4 47.6 ng/mL 1 200 9.53
5 89.4 ng/mL 1 200 17.9
6 128 ng/mL 1 200 25.6
7 0.64 ng/mL 1 200 0.13
8 1.52 ng/mL 1 200 0.304
9 1.01 ng/mL 1 200 0.203
10 1 ng/mL 1 200 0.2
11 0.81 ng/mL 1 200 0.16
12 25.3 ng/mL 1 200 5.07
13 0.58 ng/mL 1 200 0.12
14 1.87 ng/mL 1 200 0.374
15 0.81 ng/mL 1 200 0.16
16 1.19 ng/mL 1 200 0.239
17 0.58 ng/mL 1 200 0.12
18 6.17 ng/mL 1 200 1.23
19 1.17 ng/mL 1 200 0.234
20 5.2 ng/mL 1 200 1.04
21 0.95 ng/mL 1 200 0.19

Pool Samples# 1-10->Set1 Samples# 11-21->Set2[edit]

  • For Set1
  • Take 25.6ng of samples for concentration > 1ng/ul
  • Take 0.5ng for the other samples
Sample Amount ng Volume ul
1 25.60 2.49
2 25.60 3.70
3 0.50 4.17
4 25.60 2.69
5 25.60 1.43
6 25.60 1.00
7 0.50 3.85
8 0.50 1.64
9 0.50 2.46
10 0.50 2.50
JG-Microbiome-Ecoli_test-Mar22 25.60 3.07
  • Final Volume becomes 28.99ul
  • Total amount of libraries become 156.10ng
  • Final concentratioin is 5.38ng/ul = 29.50nM


  • For Set2
  • Take 5.07ng of samples for concentration > 1ng/ul
  • Take 0.2ng for the other samples
Sample Amount ng Volume ul
11 0.20 1.25
12 5.07 1.00
13 0.20 1.67
14 0.20 0.53
15 0.20 1.25
16 0.20 0.84
17 0.20 1.67
18 5.07 4.12
19 0.20 0.85
20 5.07 4.88
21 0.20 1.05
JG-Microbiome-Ecoli_test-Mar22 5.07 0.61
  • Final Volume becomes 19.72ul
  • Total amount of libraries become 21.88ng
  • Final concentratioin is 1.11ng/ul = 6.09nM