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==KAPA Library Preparation Kit illumina Platforms== ===Parts=== *Kapa Library Preparation Kit illumina platforms KR0935 V1.13 ===Samples=== *For detailed sample information, please refer to Noi's page [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:Samples_from_Moores_Cancer_Center#2014-03-04]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="background-color:#BFBFBF" align="center" | width="90" height="14" | Sample | width="61" | Tumor | width="65" | Sample | width="65" | Amount | width="65" | Qubit |style="color:#0000FF" width="65" | 100ng | width="97" | Water | width="65" | Condition |- style="background-color:#BFBFBF" align="center" | height="14" | ID | Type | DNA Type | ng | ng/ul |style="color:#FF0000" | 10ng | ul | align="center" | |- align="center" | height="14" | 6-T-1 | Colon | Tumor | align="center" | 100 | align="center" | 43.4 |style="color:#0000FF" align="center" | 2.30 | align="center" | 47.7 | Sheared |- align="center" | height="14" | 6-T-2 | Colon | Tumor | align="center" | 100 | align="center" | 31.6 |style="color:#0000FF" align="center" | 3.16 | align="center" | 46.8 | Sheared |- align="center" | height="14" | 6-T-3 | Colon | Tumor | align="center" | 100 | align="center" | 31.5 |style="color:#0000FF" align="center" | 3.17 | align="center" | 46.8 | Sheared |- align="center" | height="14" | 6-T-4 | Colon | Tumor | align="center" | 100 | align="center" | 41.7 |style="color:#0000FF" align="center" | 2.40 | align="center" | 47.6 | Sheared |- align="center" | height="14" | 6-T-5 | Colon | Tumor | align="center" | 100 | align="center" | 30.5 |style="color:#0000FF" align="center" | 3.28 | align="center" | 46.7 | Sheared |- align="center" | height="14" | 7-T-1 | Lung | Tumor | align="center" | 100 | align="center" | 30.7 |style="color:#0000FF" align="center" | 3.26 | align="center" | 46.7 | Sheared |- align="center" | height="14" | 7-T-2 | Lung | Tumor | align="center" | 100 | align="center" | 44.6 |style="color:#0000FF" align="center" | 2.24 | align="center" | 47.8 | Sheared |- align="center" | height="14" | 7-T-3 | Lung | Tumor | align="center" | 100 | align="center" | 35.9 |style="color:#0000FF" align="center" | 2.79 | align="center" | 47.2 | Sheared |- align="center" | height="14" | 7-T-4 | Lung | Tumor | align="center" | 100 | align="center" | 33.8 |style="color:#0000FF" align="center" | 2.96 | align="center" | 47.0 | Sheared |- align="center" | height="14" | 7-T-5 | Lung | Tumor | align="center" | 100 | align="center" | 20.4 |style="color:#0000FF" align="center" | 4.90 | align="center" | 45.1 | Sheared |- align="center" | height="14" | PC-T_1 | Pancreas | Tumor | align="center" | 100 | align="center" | 82.7 |style="color:#0000FF" align="center" | 1.21 | align="center" | 48.8 | Sheared |- align="center" | height="14" | PC-T_2 | Pancreas | Tumor | align="center" | 100 | align="center" | 35.8 |style="color:#0000FF" align="center" | 2.79 | align="center" | 47.2 | Sheared |- align="center" | height="14" | PC-T_4 | Pancreas | Tumor | align="center" | 100 | align="center" | 42.8 |style="color:#0000FF" align="center" | 2.34 | align="center" | 47.7 | Sheared |- align="center" | height="14" | PC-T_6 | Pancreas | Tumor | align="center" | 100 | align="center" | 15.2 |style="color:#0000FF" align="center" | 6.58 | align="center" | 43.4 | Sheared |- align="center" | height="14" | PC-T_7 | Pancreas | Tumor | align="center" | 100 | align="center" | 16.3 |style="color:#0000FF" align="center" | 6.13 | align="center" | 43.9 | Sheared |- align="center" | height="14" | NORM1-02 P1 | n/a | Cell-Free | align="center" | 10 | align="center" | 1.66 |style="color:#FF0000" align="center" | 6.02 | align="center" | 43.98 | non-Sheared |- align="center" | height="14" | NORM1-03 P1 | n/a | Cell-Free | align="center" | 10 | align="center" | 0.77 |style="color:#FF0000" align="center" | 12.99 | align="center" | 37.01 | non-Sheared |- align="center" | height="14" | COLO2-03 | n/a | Cell-Free | align="center" | 10 | align="center" | 1.11 |style="color:#FF0000" align="center" | 9.01 | align="center" | 40.99 | non-Sheared |- align="center" | height="14" | cfDNA | n/a | Cell-Free | align="center" | 10 | align="center" | 7.09 |style="color:#FF0000" align="center" | 1.41 | align="center" | 48.59 | non-Sheared |} ==Workflow== ===End Repair 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" | End Repair Master Mix: | align="center" | | align="center" | |- align="center" | height="14" | Water | align="center" | 8 | align="center" | 159.6 |- align="center" | height="14" | 10X Kapa End Repair Buffer | align="center" | 7 | align="center" | 139.65 |- align="center" | height="14" | Kapa End Repair Enzyme Mix | align="center" | 5 | align="center" | 99.75 |- align="center" |style="font-weight:bold" height="14" | Total master mix volume |style="font-weight:bold" align="center" | 20 | align="center" | 399 |} {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" | width="151" height="14" | Fragmented, double-stranded DNA | width="65" | 50ul |- align="center" | height="14" | End Repair Master Mix | 20ul |- align="center" | height="14" | Total rection Mix | 70ul |} *Mix, and incubate at 20C for 30mins *Proceed immediately to the next step ===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
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