AlanFung:LabNotes/2014/2014-9-5: Difference between revisions
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===End Repair Cleanup=== | ===End Repair Cleanup=== | ||
*Make fresh 80% ETOH100% ETOH 6384ul + H2O 1596ul | |||
*Add 120ul of diluted AMPure beads, mix by pipetting up and down (1.7X) | |||
*Incubate for 5-15mins to allow the DNA to bind to beads | |||
*Plate tubes onto a magnet to capture beads, incubate until liquid is clear | |||
*Remove and discard the supernatant | |||
*Keep the tube on the magnet, add 200l of 80% ETOH | |||
*Incubate the tube at Rt for 30sec | |||
*Remove and discard the ETOH | |||
*Repeat the washing | |||
*Allow beads to dry at RT (Do not over dry beads) | |||
*Remove tubes from magnet | |||
===A-Tailing Reaction Setup=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#BFBFBF" align="center" | |||
| width="151" height="14" | Component | |||
| width="65" | 1 Library | |||
| width="113" | 19 Libraries + 5% | |||
|- align="center" | |||
| height="14" | A-Tailing Master Mix: | |||
| align="center" | | |||
| align="center" | | |||
|- align="center" | |||
| height="14" | Water | |||
| align="center" | 25 | |||
| align="center" | 498.75 | |||
|- align="center" | |||
| height="14" | 10X Kapa A-Tailing Buffer | |||
| align="center" | 3 | |||
| align="center" | 59.85 | |||
|- align="center" | |||
| height="14" | Kapa A-Tailing Enzyme | |||
| align="center" | 2 | |||
| align="center" | 39.9 | |||
|- align="center" | |||
| height="14" | Total master mix volume | |||
| align="center" | 30 | |||
| align="center" | 598.5 | |||
|} | |||
*Resuspend the beads with 30ul master mix by pipetting up and down | |||
*Incubate at 30C for 30mins | |||
*Proceed immediately to the next step | |||
===A-Tailing Cleanup=== | |||
*Aliquot 1800l of 20%PEG/2.5M NaCl into 50ml tube | |||
*Mix 7200ul 100% ETOH with 1800ul h2o to make 80% ETOH | |||
*Add 90ul of 20%PEG/2.5M NaCl, mix by pipetting up and down | |||
*Incubate for 5-15mins to allow the DNA to bind to beads | |||
*Plate tubes onto a magnet to capture beads, incubate until liquid is clear | |||
*Remove and discard the supernatant | |||
*Keep the tube on the magnet, add 200l of 80% ETOH | |||
*Incubate the tube at Rt for 30sec | |||
*Remove and discard the ETOH | |||
*Repeat the washing | |||
*Allow beads to dry at RT (Do not over dry beads) | |||
*Remove tubes from magnet | |||
===Adapter Ligation=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#BFBFBF" align="center" | |||
| width="151" height="14" | Component | |||
| width="65" | 1 Library | |||
| width="113" | 19 Libraries + 5% | |||
|- align="center" | |||
| height="14" | Ligation Master Mix: | |||
| align="center" | | |||
| align="center" | | |||
|- align="center" | |||
| height="14" | Water | |||
| align="center" | 34 | |||
| align="center" | 678.3 | |||
|- align="center" | |||
| height="14" | 5X KAPA Ligation Buffer | |||
| align="center" | 10 | |||
| align="center" | 199.5 | |||
|- align="center" | |||
| height="14" | KAPA T4 DNA Ligase | |||
| align="center" | 5 | |||
| align="center" | 99.75 | |||
|- align="center" | |||
| height="14" | illumina Methylated Adapter | |||
| align="center" | 1 | |||
| align="center" | 19.95 | |||
|- align="center" | |||
| height="14" | Total master mix volume | |||
| align="center" | 50 | |||
| align="center" | 997.5 | |||
|} | |||
*The buffer tube ran out and I do not have enough to continue | |||
*Add 34ul water to all libraries to prevent overdrying the beads | |||
*Noi showed me another tube of buffer | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#BFBFBF" align="center" | |||
| width="151" height="14" | Component | |||
| width="65" | 1 Library | |||
| width="113" | 19 Libraries + 5% | |||
|- align="center" | |||
| height="14" | Ligation Master Mix: | |||
| align="center" | | |||
| align="center" | | |||
|- align="center" | |||
| height="14" | 5X KAPA Ligation Buffer | |||
| align="center" | 10 | |||
| align="center" | 199.5 | |||
|- align="center" | |||
| height="14" | KAPA T4 DNA Ligase | |||
| align="center" | 5 | |||
| align="center" | 99.75 | |||
|- align="center" | |||
| height="14" | illumina Methylated Adapter | |||
| align="center" | 1 | |||
| align="center" | 19.95 | |||
|- align="center" | |||
| height="14" | Total master mix volume | |||
| align="center" | 50 | |||
| align="center" | 997.5 | |||
|} | |||
*16ul of Ligation reaction/Methylation adapters mix to each reaction Mix by pipetting 10 times | |||
*Add 50ul of Ligation reaction/Methylation adapters mix, Mix by pipetting 10 times | |||
*Incubate at 20C for 15min | |||
*Add 50ul of 20%PEG/2.5M NaCl | |||
*Mix at 750rpm for 5min, 25C (lid on) | |||
*Incubate at 25C for 5 min | |||
*Put on magnet and discard spnt | |||
*Wash twice with 140ul 80% EtOH | |||
*Dry the beads | |||
*Resuspend adapter ligated DNA with ~23ul EB buffer | |||
*Store in -20C for storage |
Latest revision as of 22:29, 9 September 2014
KAPA Library Preparation Kit illumina Platforms[edit]
Parts[edit]
- Kapa Library Preparation Kit illumina platforms KR0935 V1.13
Samples[edit]
- For detailed sample information, please refer to Noi's page [[1]]
Sample | Tumor | Sample | Amount | Qubit | 100ng | Water | Condition |
ID | Type | DNA Type | ng | ng/ul | 10ng | ul | |
6-T-1 | Colon | Tumor | 100 | 43.4 | 2.30 | 47.7 | Sheared |
6-T-2 | Colon | Tumor | 100 | 31.6 | 3.16 | 46.8 | Sheared |
6-T-3 | Colon | Tumor | 100 | 31.5 | 3.17 | 46.8 | Sheared |
6-T-4 | Colon | Tumor | 100 | 41.7 | 2.40 | 47.6 | Sheared |
6-T-5 | Colon | Tumor | 100 | 30.5 | 3.28 | 46.7 | Sheared |
7-T-1 | Lung | Tumor | 100 | 30.7 | 3.26 | 46.7 | Sheared |
7-T-2 | Lung | Tumor | 100 | 44.6 | 2.24 | 47.8 | Sheared |
7-T-3 | Lung | Tumor | 100 | 35.9 | 2.79 | 47.2 | Sheared |
7-T-4 | Lung | Tumor | 100 | 33.8 | 2.96 | 47.0 | Sheared |
7-T-5 | Lung | Tumor | 100 | 20.4 | 4.90 | 45.1 | Sheared |
PC-T_1 | Pancreas | Tumor | 100 | 82.7 | 1.21 | 48.8 | Sheared |
PC-T_2 | Pancreas | Tumor | 100 | 35.8 | 2.79 | 47.2 | Sheared |
PC-T_4 | Pancreas | Tumor | 100 | 42.8 | 2.34 | 47.7 | Sheared |
PC-T_6 | Pancreas | Tumor | 100 | 15.2 | 6.58 | 43.4 | Sheared |
PC-T_7 | Pancreas | Tumor | 100 | 16.3 | 6.13 | 43.9 | Sheared |
NORM1-02 P1 | n/a | Cell-Free | 10 | 1.66 | 6.02 | 43.98 | non-Sheared |
NORM1-03 P1 | n/a | Cell-Free | 10 | 0.77 | 12.99 | 37.01 | non-Sheared |
COLO2-03 | n/a | Cell-Free | 10 | 1.11 | 9.01 | 40.99 | non-Sheared |
cfDNA | n/a | Cell-Free | 10 | 7.09 | 1.41 | 48.59 | non-Sheared |
Workflow[edit]
End Repair Reaction Setup[edit]
Component | 1 Library | 19 Libraries + 5% |
End Repair Master Mix: | ||
Water | 8 | 159.6 |
10X Kapa End Repair Buffer | 7 | 139.65 |
Kapa End Repair Enzyme Mix | 5 | 99.75 |
Total master mix volume | 20 | 399 |
Fragmented, double-stranded DNA | 50ul |
End Repair Master Mix | 20ul |
Total rection Mix | 70ul |
- Mix, and incubate at 20C for 30mins
- Proceed immediately to the next step
End Repair Cleanup[edit]
- Make fresh 80% ETOH100% ETOH 6384ul + H2O 1596ul
- Add 120ul of diluted AMPure beads, mix by pipetting up and down (1.7X)
- Incubate for 5-15mins to allow the DNA to bind to beads
- Plate tubes onto a magnet to capture beads, incubate until liquid is clear
- Remove and discard the supernatant
- Keep the tube on the magnet, add 200l of 80% ETOH
- Incubate the tube at Rt for 30sec
- Remove and discard the ETOH
- Repeat the washing
- Allow beads to dry at RT (Do not over dry beads)
- Remove tubes from magnet
A-Tailing Reaction Setup[edit]
Component | 1 Library | 19 Libraries + 5% |
A-Tailing Master Mix: | ||
Water | 25 | 498.75 |
10X Kapa A-Tailing Buffer | 3 | 59.85 |
Kapa A-Tailing Enzyme | 2 | 39.9 |
Total master mix volume | 30 | 598.5 |
- Resuspend the beads with 30ul master mix by pipetting up and down
- Incubate at 30C for 30mins
- Proceed immediately to the next step
A-Tailing Cleanup[edit]
- Aliquot 1800l of 20%PEG/2.5M NaCl into 50ml tube
- Mix 7200ul 100% ETOH with 1800ul h2o to make 80% ETOH
- Add 90ul of 20%PEG/2.5M NaCl, mix by pipetting up and down
- Incubate for 5-15mins to allow the DNA to bind to beads
- Plate tubes onto a magnet to capture beads, incubate until liquid is clear
- Remove and discard the supernatant
- Keep the tube on the magnet, add 200l of 80% ETOH
- Incubate the tube at Rt for 30sec
- Remove and discard the ETOH
- Repeat the washing
- Allow beads to dry at RT (Do not over dry beads)
- Remove tubes from magnet
Adapter Ligation[edit]
Component | 1 Library | 19 Libraries + 5% |
Ligation Master Mix: | ||
Water | 34 | 678.3 |
5X KAPA Ligation Buffer | 10 | 199.5 |
KAPA T4 DNA Ligase | 5 | 99.75 |
illumina Methylated Adapter | 1 | 19.95 |
Total master mix volume | 50 | 997.5 |
- The buffer tube ran out and I do not have enough to continue
- Add 34ul water to all libraries to prevent overdrying the beads
- Noi showed me another tube of buffer
Component | 1 Library | 19 Libraries + 5% |
Ligation Master Mix: | ||
5X KAPA Ligation Buffer | 10 | 199.5 |
KAPA T4 DNA Ligase | 5 | 99.75 |
illumina Methylated Adapter | 1 | 19.95 |
Total master mix volume | 50 | 997.5 |
- 16ul of Ligation reaction/Methylation adapters mix to each reaction Mix by pipetting 10 times
- Add 50ul of Ligation reaction/Methylation adapters mix, Mix by pipetting 10 times
- Incubate at 20C for 15min
- Add 50ul of 20%PEG/2.5M NaCl
- Mix at 750rpm for 5min, 25C (lid on)
- Incubate at 25C for 5 min
- Put on magnet and discard spnt
- Wash twice with 140ul 80% EtOH
- Dry the beads
- Resuspend adapter ligated DNA with ~23ul EB buffer
- Store in -20C for storage