Long-Range PCR on the 48.

48 Access Array IFC

PN 100-2045 B

Advanced Development Protocol

23

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Reference Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Long-Range PCR Primer Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Primer Validation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Priming the 48.48 Access Array IFC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Long-Range PCR Amplification on the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . 7 Preparing the Sample Pre-Mix and Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Loading the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Thermal Cycling the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Harvesting PCR Products from the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . . 13 Checking PCR Products on the Agilent 2100 BioAnalyzer . . . . . . . . . . . . . . . . . . . . . . . 15 For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Fluidigm

Advanced Development Protocol 23

Introduction
This document explains how to use the Fluidigm 48.48 Access Array IFC to generate Long-Range PCR products (1-10 kb). Long-Range PCR can be used to enrich specific target regions for highthroughput sequencing experiments carried out on the 454 GS FLX Sequencer, the Illumina Genome Analyzer or the Life Technologies SOLiD sequencing platform.
Design PCR Primers

Qualify Primer Sets

Select Best Primer Sets

Qualify Samples

Normalize Sample Concentrations

Load Samples and Primers on Access Array IFC

PCR amplify for 35 cycles

Recover PCR products from Access Array IFC

Qualify/Quantify PCR products

Select and Pool Samples

Clean up PCR product pool

Library Preparation

Figure 1: Long-Range PCR workflow on the Access Array system

Reference Documents
Fluidigm Access Array User Guide (PN 68000158) Fluidigm IFC Controller AX User Guide (PN 68000157) Agilent DNA 12000 Kit User Manual

Advanced Development Protocol 23

Fluidigm

Required Reagents
Stored at -20C Phusion Flash High Fidelity PCR Master Mix (New England BioLabs, PN F-548) Phusion HotStart High-Fidelity DNA Polymerase (New England BioLabs, PN F-540) 20X Access Array Loading Reagent (Fluidigm, PN 100-0883) 100 M target-specific (TS) Forward Primer Plate 100 M target-specific (TS) Reverse Primer Plate 50 ng/L genomic DNA (Coriell, PN NA17317) Stored at 4C Agilent DNA 12000 Kit Reagents (Agilent, PN 5067-1506) 1X Access Array Harvesting Reagent (Fluidigm, PN 100-1031)
cap cap

1X Access Array Long-Range PCR Hydration Reagent (Fluidigm, PN 100-1721) Stored at Room Temperature 1M Magnesium Chloride (TEKnova, PN M0300) DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) (TEKnova, PN T0221) PCR Certified Water (TEKnova, PN W3330)

Required Equipment
IFC Controller AX (2 quantity, pre- and post-PCR) 48.48 Access Array IFC(s) Fluidigm Stand-Alone Thermal Cycler (SATC) or Fluidigm FC1 Thermal Cycler MicroAmp Optical Adhesive Film (Life Technologies, PN 431197) Universal Black Microplate Lid (VWR, PN 77776-852) TempPlate Sealing Foil (USA Scientific, PN 2923-0110)

Fluidigm

Advanced Development Protocol 23

96-well plate thermal cycler Agilent 2100 BioAnalyzer Agilent DNA 12000 Chips (included in the Agilent DNA 12000 Kit) (Agilent, PN 5067-1506) 96-well plate

Long-Range PCR Primer Design Guidelines

The Long-Range PCR protocol for the Access Array system supports production of amplicons between 1 and 10 kb in size. However, within this range of product size, PCR yields will vary with the size of the amplicon (shorter fragments will produce more DNA). We strongly recommend primer sets for Long-Range PCR experiments to be designed to generate PCR products within 20% of the average PCR product size. For example, for PCR products with an average size of 5 kb, we recommend using a size range between 4500-5500 bases for the primer sets. If PCR product sizes fall outside of this range, relative coverage of each of the products in the sequencing experiment will vary considerably. 1 We recommend designing primer sets so that adjacent amplicons have an overlap of 250-500 bases. 2 Primers can be designed using the free web tool at http://flypush.imgen.bcm.tmc.edu/primer/ and the following parameters: a Primer Tm: 58C Tm 61C b Primer Size: 20 nucleotides (nt) Length 32 nt c Primer GC%: 40%<GC%<70% 3 d Max Poly-X: Avoid nucleotide runs 4 nt. Refer to the Fluidigm Access Array User Guide for additional Primer Design guidelines.

Primer Validation
IMPORTANT: The Long-Range PCR protocol for the Access Array system is only supported for primer pairs validated using the following procedure.

The target-specific (TS) Primer pairs should be validated using the protocol outlined below before running a 48.48 Access Array IFC. This protocol prepares enough reagents to perform 48 primer validation reactions in a 96-well plate. 1 Prepare the primer validation reaction components in a 1 mL tube.

Advanced Development Protocol 23

Fluidigm

Table 1: Primer Validation Reaction Mix

Component
Phusion Flash High-Fidelity PCR Master Mix (New England BioLabs, PN F-548) 20X Access Array Loading Reagent (Fluidigm, PN 100-0883) 1M Magnesium Chloride (TEKnova, PN M0300) Phusion HotStart High-Fidelity DNA Polymerase (New England BioLabs, F-540) 50 ng/L genomic DNA Total cap

Volume per Reaction (L)

5.0 0.485

Volume for 60 Reactions (L)

300.0 29.1

0.015 0.50 4.0 10

0.9 30.0 240.0 600

Vortex the Primer Validation Reaction Mix for a minimum of 20 seconds, and centrifuge for at least 30 seconds to spin down all components.
NOTE: The volumes provided in the table above are sufficient for 60 11 L reactions in a 96-well plate. We recommend preparing this amount to provide sufficient reagent to minimize errors due to pipetting.

Set up the PCR Reactions in a 96-well PCR plate. a Add 10.0 L of Primer Validation Reaction Mix to each well. b Add 0.5 L of the TS Forward Primer for 10 M final concentration. c Add 0.5 L of the TS Reverse Primer for 10 M final concentration. The total PCR reaction volume is 11 L.

4 5 6

Tightly seal the plate with a TempPlate Sealing Foil (USA Scientific, PN 2923-0110) to minimize dehydration during thermal cycling. Vortex the 96-well PCR reaction plate for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components. Run the PCR Reactions. d Load the 96-well plate onto the thermal cycler. e Run the PCR protocol described below:

Fluidigm

Advanced Development Protocol 23

Table 2: Long-Range PCR protocol for 1-10 kb amplicons

Step

Temperature
50 C 70C 98C 98C 61C 72C Go to Step 4, repeat 34 times 72C 4C

Time
2 min 12 min 10 sec 1 sec 5 sec 5 min

1 2 3 4 5 6 7 8 9 7

10 min

Check PCR Products on the Agilent 2100 BioAnalyzer. a Dilute 2 L of each PCR product with 10 L of PCR Certified Water (PN W3330). b Run 1 L of the diluted PCR product from each of the PCR reactions on an Agilent DNA 12000 chip on the Agilent 2100 BioAnalyzer. Follow the Agilent DNA 12000 Kit User Manual for details. c Inspect the results from the BioAnalyzer chip run. Please refer to the Fluidigm Access Array User Guide for details on checking PCR products on the Agilent 2100 BioAnalyzer. Successful reactions will produce a single band of the expected size. If the PCR reactions contain multiple bands, or bands of incorrect size, the primer pair should be removed from the test set. In these cases, check the amplicon sequence for any problems such as a high G/C content or long nucleotide repeats. If there are no apparent problems with the amplicon sequence, redesign the primers by moving the primer location upstream or downstream by 1000-2000 bp or by splitting the target region into two amplicons.

Select the TS Primer pairs that produce the correct PCR products and continue with the 48.48 Access Array IFC.

Priming the 48.48 Access Array IFC

1 Inject control line fluid into both IFC accumulators.
NOTE: Control Line Fluid on the IFC or in the inlets makes the IFC unusable. Use only 48.48 syringes with 300 L of Control Line Fluid (PN 89000020).

Add 500 L of 1X Access Array Harvest Reagent (Fluidigm, PN 100-1031) into the H1-H3 wells on the IFC.

Advanced Development Protocol 23

Fluidigm

Add 500 L of 1X Access Array Long-Range PCR Hydration Reagent (Fluidigm, PN1001721) into the H4 well on the IFC.
NOTE: Make sure the H1, H2 and H3 wells are loaded with Access Array Harvest Reagent (Clear) and the H4 well is loaded with Access Array Hydration Reagent.

H1

H2

Harvest Reagent

H3

H4

Hydration Reagent

Figure 2: Loading positions of the Harvest Reagent and Hydration Reagent on the 48.48 Access Array IFC

Place the 48.48 Access Array IFC into the Pre-PCR IFC Controller AX located in the PrePCR Lab and run Prime (151x) script.
NOTE: Load the IFC in the Pre-PCR IFC Controller AX in the Pre-PCR Lab within 60 minutes of priming.

Long-Range PCR Amplification on the 48.48 Access Array IFC

Prepare the 20X Primer Solutions
1 Take the 100 M target-specific (TS) Forward and Reverse Primer plates out of the freezer and warm the plates up to room temperature. Vortex the 20X Primer solutions for a minimum of twenty seconds, and centrifuge for 30 seconds to spin down all components. In a DNA-free hood, prepare the 20X Primer solution for each primer pair in a 96-well plate as shown below in Table 3.

Fluidigm

Advanced Development Protocol 23

Table 3: 20X Primer solutions

Component
TS Forward Primer (100 M) TS Reverse Primer (100 M) 20X Access Array Loading Reagent (Fluidigm, PN 100-0883) DNA Suspension Buffer Total cap

Volume (L)
0.5 0.5 0.25 3.75 5

Vortex the 20X Primer solution plate for 20 seconds, and centrifuge for 30 seconds to spin down all components. Store the plate at 4C.
NOTE: To avoid multiple freeze-thaws of primers, a larger volume of the 20X Primer solution can be prepared and aliquoted into multiple 96-well PCR plates. Store the unused 20X Primer solution plates at -20C.

NOTE: The final TS Forward and Reverse Primer concentrations are 10 M in the 20X Primer solution. The final TS Forward and Reverse Primer concentrations in the 48.48 Access Array IFC reaction chambers are 500 nM.

Preparing the Sample Pre-Mix and Samples

Prepare the Sample Pre-Mix Solution
1 2 Normalize the concentration of each genomic DNA sample to 50 ng/L. In a DNA-free hood, combine the components listed in Table 4 below. This protocol prepares enough Sample Pre-Mix solution for 60 reactions. This is enough reagent to load one 48.48 Access Array IFC with 16 additional reactions to compensate for dead volume and pipetting error.

Advanced Development Protocol 23

Fluidigm

Table 4: Sample Pre-Mix solution

Component
Phusion Flash High-Fidelity PCR Master Mix (New England BioLabs, PN F-548) 20X Access Array Loading Reagent (Fluidigm, PN 100-0883) cap

Volume per Reaction (L)

2.5 0.242

Volume per 60 Reactions (L)

150.0 14.52

1M Magnesium Chloride (Teknova, PN M0300) Phusion HotStart High-Fidelity DNA Polymerase (New England BioLabs, F-540) Total

0.008 0.25 3

0.48 15.0 180

Vortex the tube for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components. Combine the components listed below in a 96-well plate to prepare 48 individual Sample Mix solutions.

Prepare the Sample Mix Solutions

1

Table 5: Sample Mix solutions

Component
Sample Pre-Mix 50 ng/L genomic DNA Total

Volume (L)
3.0 2.0 5

Vortex the Sample Mix solutions for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components.

NOTE: It is essential that you vortex all components to ensure complete mixing.

Loading the 48.48 Access Array IFC

1 2 Pipette 4 L of 20X Primer solution into each of the primer inlets. Pipette 4 L of Sample Mix solution into each of the sample inlets.

Fluidigm

Advanced Development Protocol 23

Sample Mix

Primer Mix

H1

H2

H3

H4

Figure 3: 48.48 Access Array IFC Sample and Assay inlet pipetting map

10

Advanced Development Protocol 23

Fluidigm

NOTE: We recommend using an 8-channel pipette to load the Sample Mix solution and 20X Primer solution. The recommended pipetting order is shown below.

Step 1
1 4 2 3 6 9 5 7 8

Step 2
1 4 2 3 6 9 5 7 8

Step 3
1 4 2 5 3 6 9

Step 4
1 4 2 3 6 9 5 7 8

Step 5
1 4 2 5 3 6 9

Step 6
1 4 2 5 3 6 9

7 8

7 8

7 8

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Figure 4: 48.48 Access Array IFC pipetting scheme

3 4

Load the 48.48 Access Array IFC into the Pre-PCR IFC Controller AX in the Pre-PCR Lab and run Load Mix (151x) script. When the script is finished, press Eject to remove the 48.48 Access Array IFC from the Pre-PCR IFC Controller AX.

Thermal Cycling the 48.48 Access Array IFC

1 2 Check the top surface of the 48.48 Access Array IFC chip for any large particles or debris. Remove the particles gently with scotch tape. Cut one of the MicroAmp Optical Adhesive Films (Life Technologies, PN 431197) into a 1.7 inch square using the Template for cutting the PCR Film shown in Figure 4. This will be used as the pre-cut PCR film for thermal cycling.

11

Fluidigm

Advanced Development Protocol 23

Place the film on the surface of the chip starting at one corner, smoothing the film gradually to avoid capturing air bubbles. Make sure to remove any air bubbles between the PCR film and the chip.
A B

1.7

1.7

Template for cutting the PCR film

Figure 5A: Placement of the pre-cut PCR film onto the 48.48 Access Array IFC chip Figure 5B: Template for cutting the PCR film

4 5 6

Remove the blue IHS protector from the bottom of the 48.48 Access Array IFC. Place the 48.48 Access Array IFC on the Thermal Cycler. Place the Universal Microplate Lid (PN 77776-852) on top of the 48.48 Access Array IFC carrier as shown in Figure 5.

Figure 6: Placement of the Universal Microplate Lid onto the 48.48 Access Array IFC carrier

12

Advanced Development Protocol 23

Fluidigm

7 8

Turn on the vacuum. Select the AALR48V1 PCR Protocol listed in Table 6 (for the SATC) or AA 48x48 Long Range v1 (for the FC1), and follow the Thermal Cycler instructions in the Fluidigm Access Array User Guide. Press START to begin PCR.

Table 6: AALR48V1 - Long-Range PCR protocol for 2-10 kb amplicons

Step

Temperature
50 C 70C 98C 98C 61C 72C Go to Step 4, repeat 34 times 72C 4C

Time
2 min 12 min 10 sec 1 sec 5 sec 5 min

1 2 3 4 5 6 7 8 9

10 min Hold at 4C

Harvesting PCR Products from the 48.48 Access Array IFC

1 2 3 4 5 6 7 After the PCR has finished, press ENTER and turn off the vacuum on the Fluidigm Thermal Cycler. Move the 48.48 Access Array IFC into the Post-PCR Lab for harvesting. Remove the Universal Microplate Lid (PN 77776-852) from the 48.48 Access Array IFC. Remove the remaining 1X Access Array Harvest Reagent from the H1-H3 wells. Remove the remaining 1X Access Array Hydration Reagent from the H4 well. Pipette 600 L of fresh 1X Access Array Harvest Reagent into each of the H1-H4 wells. Pipette 2 L of 1X Access Array Harvest Reagent into each of the sample inlets on the IFC.

13

Fluidigm

Advanced Development Protocol 23

2 L/inlet Harvest Reagent

600 L Harvest Reagent

H1

H2

H3

H4

600 L Harvest Reagent

Figure 7: Harvest Reagent loading positions on Post-PCR 48.48 Access Array IFC

8 9

Place the 48.48 Access Array IFC into the Post-PCR IFC Controller AX located in the Post-PCR Lab and run Harvest (151x) script. When the Harvest (151x) script has finished, remove the 48.48 Access Array IFC from the Post- PCR IFC Controller.

10 Label a 96-well plate with the 48.48 Access Array IFC barcode. Carefully transfer the harvested PCR products from each of the sample inlets into columns 1-6 of the 96-well PCR plate. 11 Store the PCR products at -20C.
NOTE: Remove PCR products from the 48.48 Access Array IFC using an 8-channel pipette in the same order as you loaded the IFC.

14

Advanced Development Protocol 23

Fluidigm

Figure 8: PCR product transfer map from the 48.48 Access Array IFC to a 96-well PCR plate

Checking PCR Products on the Agilent 2100 BioAnalyzer

1 Use the Agilent DNA 1200 chips from the Agilent DNA 1200 Kit to check 1 L of PCR product from each of the PCR reactions described above. Follow the Agilent DNA 1200 Kit User Manual for details. Check the PCR product pool to confirm that products of the correct size have been generated. The product pool contains a combination of all 48 PCR products generated by the Access Array IFC, and will appear as a smear on the BioAnalyzer. A histogram of expected PCR product sizes can be used for comparison with the electropherogram trace from the PCR product pool. Store the PCR products at -20C. Refer to the Fluidigm Access Array User Guide for more information about this workflow.

3 4

15

Fluidigm

Advanced Development Protocol 23

For More Information

To find out more about the information in this or any other Fluidigm Advanced Development Protocol, contact techsupport@fluidigm.com or call: North America: 1 866 358 4354 Europe: +33 1 60 92 42 40 Japan: +81 3 3555 2351 All other countries: +1 650 266 6100

Copyright, Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, BioMark, Access Array, Dynamic Array, Digital Array, FC1, Topaz, and NanoFlex are trademarks or registered trademarks of Fluidigm Corporation. All other trademarks are the property of their respective owners. Part Number 100-2045 B

16