AlanFung:LabNotes/2014/2014-9-5: Difference between revisions

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===Parts===
===Parts===
*Kapa Library Preparation Kit illumina platforms KR0935 V1.13
*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" | &nbsp;
|-  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==
==Workflow==
===End Repair Reaction Setup===
===End Repair Reaction Setup===
Line 8: Line 223:
| width="151" height="14" | Component
| width="151" height="14" | Component
| width="65" | 1 Library
| width="65" | 1 Library
| width="113" | 15 Libraries + 5%
| width="113" | 19 Libraries + 5%


|-  align="center"
|-  align="center"
Line 18: Line 233:
| height="14" | Water
| height="14" | Water
| align="center" | 8
| align="center" | 8
| align="center" | 126
| align="center" | 159.6


|-  align="center"
|-  align="center"
| height="14" | 10X Kapa End Repair Buffer
| height="14" | 10X Kapa End Repair Buffer
| align="center" | 7
| align="center" | 7
| align="center" | 110.25
| align="center" | 139.65


|-  align="center"
|-  align="center"
| height="14" | Kapa End Repair Enzyme Mix
| height="14" | Kapa End Repair Enzyme Mix
| align="center" | 5
| align="center" | 5
| align="center" | 78.75
| align="center" | 99.75


|- style="font-weight:bold" align="center"
|- align="center"
| height="14" | Total master mix volume
|style="font-weight:bold" height="14" | Total master mix volume
| align="center" | 20
|style="font-weight:bold" align="center" | 20
| align="center" | 315
| align="center" | 399


|}
|}
Line 54: Line 269:


===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" | &nbsp;
| align="center" | &nbsp;
|-  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" | &nbsp;
| align="center" | &nbsp;
|-  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" | &nbsp;
| align="center" | &nbsp;
|-  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