Revision as of 16:22, 9 April 2011 by >Alan6017518
Library Construction on sample 1-21 from Dr. Heller
Qubit Quantification
- 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 |
|
Concentrate Samples using speed vacuum centrifuge concentrator
- Dry sample with speedvac.
- Add 5ul ddH2Oto dry pellet
Low input transposase-based library preparation (Shendure's Protocol) for the low amount samples
- 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
Cycle under standard Nextera conditions for 20 cycles
Clean up using Minelut,elute in 20ul EB
Tagmentation Reaction for samples with high concentration
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
- 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
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
- 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
File:040511 P3.jpg
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
Qubit Quantification
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
- 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