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Rapid Library Preparation Method Manual GS FLX Titanium Series October 2009 (Rev. Jan 2010)
Rapid Library Preparation Method ManualGS FLX Titanium Series
October 2009 (Rev. Jan 2010)
2 / 8 October 2009 (Rev. Jan 2010)
Rapid Library Preparation Method Manual
Workfl ow1.
Preparing a Rapid library consists of 6 major steps, outlined in the workfl ow in Figure 1.
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Figure 1: Workfl ow of the Rapid Library Preparation.
Two procedures are described here. The Individual Sample Cleanup (ISC) is shown in black type while the Parallel Sample Cleanup (PSC), when different, is shown in blue type. ISC is for handling samples in 1.7 ml microcentrifuge tubes; while PSC is for handling samples in 200 µl PCR tubes, and is amenable for automation. When processing 8 samples or more, we recommend using the PSC workfl ow and Agencourt’s 96 ring Magnetic Particle Concentrator and 96-well plates recommended by Agencourt.
The procedure presented here is for one sample, processed either in ISC or PSC.
Sample Requirements2.
Sample DNA should be:
� double-stranded
� OD260/280
≥ 1.8
� concentration ≥ 5 ng/µl
� fragment size > 1.5 kb
Procedure3.
Room temperature is +22 to +25°C.
DNA Fragmentation by Nebulization3.1
1 Start with 500 ng of sample DNA in a 1.7 ml microcentrifuge tube.
2 Add TE Buffer to a fi nal volume of 100 µl.
3 Using sterile gloves, affi x a Nebulizer Condensor tube around the Aspiration tube. To ensure proper function, make sure to push the Condensor tube all the way down around the base of the Aspiration tube, being careful not to rotate the Aspiration tube, and press the vented cap into the Nebulizer top (Figure 2A).
4 Set the assembled Nebulizer top, with the aspiration tube pointing upwards; making sure that the inside parts do not contact any contaminated surfaces (counter top, hands).
5 Pipet the 100 µl DNA sample in the Nebulizer cup.
6 Add 500 µl of Nebulization Buffer, pipet up and down to mix.
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Workfl ow
For life science research only. Not for diagnostic procedures.
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Rapid Library Preparation Method Manual
7 Screw the Nebulizer top to the cup, transfer the cup to the external vented hood and connect the tubing to the nitrogen tank (Figure 2B).
A B
Figure 2: Assembling the Nebulizer.
8 Apply 30 psi (2.1 bar) of nitrogen for 1 minute.
9 Disconnect the tubing and remove the cup from the hood.
10 Remove the Nebulizer top from the cup.
11 Add 2.5 ml of PBI Buffer.
12 Pipet up and down to mix.
13 Purify the nebulized DNA sample on a column from the Qiagen MinElute PCR Purifi cation kit, as follows, with all centrifugation steps carried out at 13,000 rpm in a tabletop centrifuge:
a. Load 750 µl of the nebulized DNA at a time into a single column.
b. Centrifuge for 15 seconds and discard the fl ow-through.
c. Repeat steps a and b three more times, using the same column.
d. Centrifuge for 1 minute. Discard all the fl ow-through.
e. Add 750 µl of PE Buffer, centrifuge for 1 minute, and discard the fl ow-through.
f. Centrifuge 1 minute, rotate the column 180º, centrifuge 1 minute.
g. Elute in new tube with 16 µl of TE Buffer by centrifuging for 1 minute.
h. Transfer the sample to a 200 µl PCR tube.
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Fragment End Repair3.2
1 In a 1.7 ml microcentrifuge tube, prepare the End Repair mix, as follows.
2.5 µl RL 10× PNK Buffer 2.5 µl RL ATP 1 µl RL dNTP 1 µl RL T4 Polymerase 1 µl RL PNK 1 µl RL Taq Polymerase 9 µl Total volume
2 Pipet up and down to mix, and add the 9 µl of End Repair mix to the DNA sample:
3 Vortex for 5 seconds, then spin for 2 seconds in a mini centrifuge.
4 Run the End Repair program on a thermocycler, with the heated lid on:
25ºC for 20 min 72ºC for 20 min 4ºC on hold
5 While the program is running, you can prepare the Agencourt AMPure beads as described in Section 3.3, below. You will use them in Section 3.5.
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Procedure
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Rapid Library Preparation Method Manual
AMPure Bead Preparation3.3
1 Vortex the AMPure bead bottle for 20 seconds, or until the beads are completely resuspended.
2 Aliquot 125 µl of AMPure beads in a 1.7 ml microcentrifuge tube (ISC) or in a 200 µl PCR tube (PSC).
3 Place the tube on the Magnetic Particle Concentrator (MPC) (ISC) or a 96 ring MPC (PSC).
4 When the beads have completely pelleted on the side of the tube, carefully remove and discard all supernatant, without disturbing the beads.
5 For ISC only: add 73 µl of TE Buffer to the beads and vortex 5 seconds.
6 Add 500 µl (ISC) or 125 µl (PSC) of Sizing Solution to the beads, vortex for 5 seconds or pipet up and down 10 times (PSC), and spin for 2 seconds in a mini centrifuge.
7 Keep the tube on ice, until you reached Section 3.5.
8 Prepare 5 ml of 70% ethanol, by adding 3.5 ml of 100% ethanol to 1.5 ml Molecular Biology Grade Water, and vortex. You will use this 70% ethanol solution in Section 3.5.
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Adaptor Ligation3.4
1 Once the End Repair program has completed from Section 3.2, add 1 µl of RL Adaptor or of RL MID Adaptor to the reaction tube.
2 Add 1 µl of RL Ligase to the reaction tube.
3 Vortex 5 seconds, then centrifuge for 2 seconds in a mini centrifuge.
4 Incubate at 25ºC for 10 minutes.
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Small Fragment Removal3.5
1 Add the sample to the AMPure beads prepared in Section 3.3. Vortex for 5 seconds and spin for 2 seconds in a mini centrifuge.
2 Incubate at room temperature for 5 minutes.
3 Place the tube on the MPC.
4 When the beads have fully pelletted on the wall of the tube, carefully remove and discard the supernatant.
5 Add 100 µl (ISC) or 25 µl (PSC) of TE Buffer. Vortex for 5 seconds (ISC) or pipet up and down 10 times (PSC).
6 Add 500 µl (ISC) or 125 µl (PSC) of Sizing Solution. Vortex for 5 seconds (ISC) or pipet up and down 10 times (PSC).
7 Incubate at room temperature for 5 minutes.
8 Place the tube on the MPC.
9 When the beads have fully pelleted on the wall of the tube, carefully remove and discard the supernatant.
10 Repeat steps 5 to 9, once.
11 Keeping the tube on the MPC, wash the beads twice, as follows:
a. Add 1 ml (ISC) or 175 µl (PSC) of 70% ethanol.
b. Completely remove and discard the ethanol.
12 Keeping the tube on the MPC, uncap the tube and air dry the pellet at room temperature for 2 minutes.
13 Remove the tube from the MPC.
14 Add 53 µl of TE Buffer. Vortex for 5 seconds and spin for 2 seconds in a mini centrifuge.
15 Place the tube on the MPC, wait for the beads to pellet on the wall of the tube and transfer 50 µl of the SUPERNATANT, containing the library, to a new, labeled 1.7 ml microcentrifuge tube. Make sure not to carry-over any beads in this process as they will cause incorrect readings during library quantitation.
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Procedure
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Rapid Library Preparation Method Manual
Library Quantitation3.6
Use either a single cuvette or a 96-well plate fl uorometer to quantitate the DNA library. We recommend the TBS 380 Fluorometer (Turner Biosystems) for single use cuvette.
Preparing the Standard3.6.1
When using the 96-well plate fl uorometer (Section 3.6.3), prepare twice the volume for each standard dilution because you will need to read the standards in duplicate.
To generate the standard curve, begin by labeling 8 tubes, 1 to 8.
1 In tube 1, prepare a 2.5 × 109 molecule/µl solution of the RL Standard by mixing 90 µl of the RL Standard (orange cap) with 90 µl of TE Buffer.
2 Fill the remaining 7 tubes (tubes 2 to 8) with 60 µl of TE Buffer.
3 Transfer 120 µl from tube 1 into tube 2.
4 Vortex for 5 seconds and spin for 2 seconds in a mini centrifuge.
5 Change pipet tip and transfer 120 µl of tube 2 into tube 3.
6 Vortex for 5 seconds and spin for 2 seconds in a mini centrifuge.
7 Proceed with the same serial dilution (transferring 120 µl of one tube into the next, vortexing for 5 seconds, and changing pipet tip between each dilution) for the remaining 5 tubes.
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Using the TBS 380 Fluorometer3.6.2
1 Transfer 50 µl of the 8 dilutions of the RL Standard into 8 cuvettes.
2 To generate a blank, transfer 50 µl of TE Buffer in a cuvette.
3 Set the TBS 380 fl uorometer on the Blue channel with the Blue cuvette holder insert. Set the standard value (Std Val) to 250.
4 Calibrate the fl uorometer with the blank and the 2.5 × 109 molecule/µl solution RL Standard.
5 Read and record the relative fl uorescence units (RFU) of each dilution.
6 Transfer 50 µl of the sample library in a cuvette. Read and record the RFU. DO NOT DISCARD THE SAMPLE.
7 Transfer the sample library back to its tube with a 20 µl pipet tip.
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Using a 96-well Plate Fluorometer3.6.3
When using a 96-well plate fl uorometer, we recommend reading fl uorometry values in the Costar half-area 96-well plates. Enter an excitation value of 465 nm, and an emission value of 515 nm.
1 Prepare twice the volume of each standard dilution and distribute on the 96-well plate, in duplicate wells, 50 µl of each standard.
2 Transfer 50 µl of the sample library into an individual well.
3 Read and record relative fl uorescence units (RFU) for the sample library. DO NOT DISCARD THE SAMPLE.
4 Transfer the sample library back to its tube.
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Procedure
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Rapid Library Preparation Method Manual
Generating a RL Standard Curve and Calculating the Sample Concentration3.6.4
To generate a standard curve of fl uorescence readings and calculate the library sample concentration, use either the Rapid Library Quantitation Calculator, or Excel software, selecting XY (scatter), creating a linear trend line, and selecting for the mathematical equation of the linear regression. An example of a RL Standard Curve is in Section 4.1. It is also possible to use software supplied by your fl uorometer manufacturer to generate a standard curve and calculate the concentration of the sample.
Using the Rapid Library Quantitation Calculator3.6.4.1
Go to the Web page www.454.com/my454 to access the Rapid Library Quantitation Calculator. Explanations on how to use the Calculator can be found on the web page.
Using Excel Software3.6.4.2
1 Create an XY (scatter) plot in Excel using the fl uorescence readings as the x axis and the RL Standard concentrations (molecules/µl) as the y axis.
2 Right click any of the data points in the plot and select add trend line.
3 Set regression type to linear.
4 Select options tab then choose display equation on chart as well as display R-squared value on chart.
5 R2, the correlation coeffi cient for the linear regression, must have a minimum value of 0.9.
6 If the sample RFU falls above the standard curve, dilute the sample by adding 50 µl of TE Buffer and read the RFU again.
7 The total DNA library amount should be at minimum 7.3 × 109 molecules which corresponds to a concentration of ≥1.46 × 108 mol/µl.
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The serial dilution of the RL Standard produced the following concentrations:
Dilution tube number (dilution factor) RL Standard (mol/µl)
1 (no dilution) 2.50 × 109
2 (2/3rd dilution of tube 1) 1.67 × 109
3 (2/3rd dilution of tube 2) 1.11 × 109
4 (2/3rd dilution of tube 3) 7.41 × 108
5 (2/3rd dilution of tube 4) 4.94 × 108
6 (2/3rd dilution of tube 5) 3.29 × 108
7 (2/3rd dilution of tube 6) 2.19 × 108
8 (2/3rd dilution of tube 7) 1.46 × 108
Library Quality Assessment3.7
1 Run a 1 µl aliquot of the DNA library on an Agilent Bioanalyzer High Sensitivity DNA chip, to assess the quality of the library. See Section 4.2 for a representative trace of a sample library run on the High Sensitivity Chip.
2 Assess the quality of the DNA library for the characteristics listed below:
Library Characteristics Expected Results
Average fragment length Between 600 bp and 900 bp
Lower size cut-off <10% below 350 bp
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Preparing Working Aliquots3.8
1 Dilute an aliquot of the DNA library to a working stock of 1 × 107 molecules/µl, in TE Buffer.
2 Transfer the working stock in 25–100 µl aliquots, and store these at -15 to -25ºC, for up to 2 months.
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Immediately before emPCR amplifi cation, heat denature the Rapid library sample at 95°C for 2 minutes, using a thermocycler with heated lid on.
Procedure
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Rapid Library Preparation Method Manual
Appendix4.
Example of Standard Curve for the RL Standard4.1
Rapid Library Standard Curve
Figure 3: Standard curve using Excel software.
Example of a Library Sample Run on a Bioanalyzer High Sensitivity Chip4.2
Figure 4: High Sensitivity Chip profi le of a Rapid library sample.
Procedure
Published by
Roche Diagnostics GmbHRoche Applied Science68298 MannheimGermany
© 2010 Roche Diagnostics All rights reserved.
�0110
For Life Science Research Only. Not for Use in Diagnostic Procedures
454, 454 LIFE SCIENCES, 454 SEQUENCING, GS FLX, GS FLX TITANIUM,
emPCR, PICOTITERPLATE, and PTP are trademarks of Roche.
Other brands or product names are trademarks of their respective holders.
Rapid Library Preparation Method Manual
Table of Contents
1. Workfl ow ...........................................................................................................................2
2. Sample Requirements ...............................................................................................2
3. Procedure ..........................................................................................................................2
3.1 DNA Fragmentation by Nebulization .......................................................................................2
3.2 Fragment End Repair ......................................................................................................................3
3.3 AMPure Bead Preparation ...........................................................................................................4
3.4 Adaptor Ligation ...............................................................................................................................4
3.5 Small Fragment Removal ..............................................................................................................4
3.6 Library Quantitation ........................................................................................................................5
3.6.1 Preparing the Standard .................................................................................................................53.6.2 Using the TBS 380 Fluorometer .................................................................................................53.6.3 Using a 96-well Plate Fluorometer ............................................................................................53.6.4 Generating a RL Standard Curve and Calculating the Sample Concentration ........6
3.7 Library Quality Assessment ..........................................................................................................6
3.8 Preparing Working Aliquots .........................................................................................................6
4. Appendix ...........................................................................................................................7
4.1 Example of Standard Curve for the RL Standard ................................................................7
4.2 Example of a Library Sample Run on a Bioanalyzer High Sensitivity Chip ................7
