Calendar/2011-3-28: Difference between revisions
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>Alan6017518 |
>Alan6017518 |
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*Final Volume becomes 28.99ul | *Final Volume becomes 28.99ul | ||
*Total amount of libraries become 156.10ng | *Total amount of libraries become 156.10ng | ||
*Final concentratioin is 5.38ng/ul | *Final concentratioin is 5.38ng/ul = 29.50nM | ||
Line 297: | Line 297: | ||
*Final Volume becomes 19.72ul | *Final Volume becomes 19.72ul | ||
*Total amount of libraries become 21.88ng | *Total amount of libraries become 21.88ng | ||
*Final concentratioin is 1.11ng/ul | *Final concentratioin is 1.11ng/ul = 6.09nM |
Revision as of 16:22, 9 April 2011
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 |
- Samples are too dilute
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
- libraries for low starting amount didn't amplify (Cycle1-20)
- Added 5 more cycles
- 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
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