Calendar/2011-3-28
Jump to navigation
Jump to search
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
- 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[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