Agilent 165kb BAC Kit Analyzer Quick Guide PDF

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Agilent 165 kb BAC Kit Quick Guide for the Femto Pulse System

The Agilent Femto Pulse system is an automated capillary electrophoresis platform for scalable, flexible, fast, and reliable electrophoresis of nucleic acids.

This Quick Guide is intended for use with the Agilent Femto Pulse system only. The 165 kb BAC kit (275 samples) (Part # FP-1004-0275) is designed for the Pulse-Field CE separation and sizing of bacterial artificial chromosome (BAC) fragment up to 165 kB in length.

Specifications

Analytical specifications1 165 Kb BAC assay

Sizing Range 75 bp 165 kb

Sizing Accuracy1 + 15% or better

Sizing Precision1 15% CV

DNA Fragment Concentration Range

Single Fragment 48,500 bp 1.6 pg/L 25 pg/L input DNA

Single Fragment 48,500 bp 3 pg/L 50 pg/L input DNA

Multiple Fragments 12.5 pg/L 100 pg/L input DNA

Physical Specifications

Total electrophoresis run time 170 minutes

Samples per run 12-Capillary: 11 (+1 Ladder Well)

Sample volume required 2 L

Kit stability 4 months

1 Result based on sample BAC fragments.

FP-1004 165 kb BAC Quick Guide for the Femto Pulse System

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Kit Components 275 Sample Kit

Kit Component Number

Part Number

(Re-order Number)

Description Quantity Per Kit

5191-6604* Femto Pulse, 4oC

FP-5001-0250 FP Large DNA Separation Gel, 250 mL 1

DNF-306-0005 BF-P25 Blank Solution, 5 mL 1

DNF-325-0075 5x Inlet Buffer, 75 mL 1

DNF-498-0012 Dilution Buffer 0.25x TE, 12 mL 1

DNF-497-0060 0.25x TE Rinse Buffer, 60 mL 1

FP-1004-FR* 165 kb BAC, FR

FP-6001-U030 FP Intercalating Dye, 30 L 1

FP-8001-0003 FP gDNA Diluent Marker, 3 mL 3

FP-7004-U035 FP 165 kb BAC Ladder, 35 L 1

FP-8004-U100 FP 165 kb Upper Marker, 100 L 1

5191-6618* BAC, RT

C27-130 Eppendorf LoBind 0.5 mLTubes (Bag of 50) 1

C280-101 Wide-Bore Genomic Pipette Tips, 1 Box 1

DNF-425-0050 5x Conditioning Solution, 50 mL 1

GP-435-0100 Storage Solution, 100 mL 1

FS-SMO15 Mineral Oil Dropper Bottle, 15 mL 1

*Not Orderable.

WARNING Refer to product safety data sheets for further information

When working with the Femto Pulse assay follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

FP-1004 165 kb BAC Quick Guide for the Femto Pulse System

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Additional Material Required for Analysis with the Femto Pulse System

Femto Pulse system with LED fluorescence detection:

Femto Pulse system (p/n M5330AA)

FP 12-Capillary Array, 22 cm (p/n A1600-1250-2240)

Agilent Femto Pulse controller software (Version 1.0 or higher)

Agilent ProSize data analysis software (Version 3.0 or higher)

Additional equipment/reagents required (not supplied)

96-well PCR sample plates. Please refer to Appendix Femto Pulse Compatible Plates and Tubes in the Femto Pulse

System User Manual for a complete approved sample plate list

Multichannel pipettor(s) and/or liquid handling device capable of dispensing 1 100 L volumes (sample plates) and

1,000 L volumes (inlet buffer plate)

Pipette tips

Wide-Bore Genomic pipette tips, Thermo Scientific Part #21-402-157 (as needed for pipetting gDNA samples)

96-well plate centrifuge (for spinning down bubbles from sample plates)

Sub-micron filtered DI water system (for diluting the 5x Inlet Buffer and 5x Conditioning Solution)

96-deepwell 1mL plate: Fisher Scientific #12-566-120 (inlet buffer and/or waste plate)

Reagent reservoir, 50 mL (VWR #89094-680 or similar) (for use in pipetting inlet buffer plates/sample trays)

Conical centrifuge tubes for prepared separation gel/dye mixture and/or 1x Conditioning Solution

50 mL (Femto Pulse system): BD Falcon #352070, available from Fisher Scientific #14-432-22 or VWR #21008-940

250 mL (Femto Pulse system or larger volumes): Corning #430776, available from Fisher Scientific #05-538-53 or

VWR #21008-771

Essential Measurement Practices

Environmental conditions

Ambient operating temperature: 19 25 C (66 77 F)

Keep reagents during sample preparation at room temperature

Steps before sample preparation

Allow Reagents to equilibrate at room temperature for 30 min prior to use

Store the gDNA Diluent Marker solution, 165 kb Upper Marker and 165 kb BAC

Ladder at -20C upon arrival.

Pipetting practice

Pipette reagents carefully against the side of the 96-well sample plate or sample

tube

Ensure that no sample or Diluent Marker remains within or on the outside of the tip

Use only wide bore pipette tips (provided) to aliquot and mix the 165 kb Ladder and

165 kb Upper Marker.

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Marker/Ladder/Sample Preparation

165 kb Upper Marker Handling and Storage

Prior to first use, the 165 kb Upper Marker should be aliquoted to minimize the number of freeze/thaw cycles.

1. Equilibrate the 165 kb Upper Marker to room temperature for about 30 min and mix by gently pipetting up-and-down up to 3 times with wide-bore genomic pipette tips (provided with kit) and a pipettor set to a 15 L volume. Do not vortex of flick the Marker tube. Only wide-bore genomic pipette tips should be used to mix the 165 kb Upper Marker solution.

2. Aliquot the 165 kb Upper Marker solution into 7 aliquots, 14 L each, using the provided wide-bore genomic tips and the provided Eppendorf LoBind 0.5 mL tubes. Each aliquot is good for 4-times use (3 L per use).

3. Store the 165 kb Upper Marker aliquots at -20C. Store the in-use vial of the 165 kb Upper Marker at 2-8C for up to 2 weeks. Avoid freeze-thawing of the 165 kb Upper Marker more than 4 times (more frequent freeze-thawing may result in degradation of the 165 kb Upper Marker).

165 kb Upper Marker Working Solution Preparation

1. Before use, equilibrate the 165 kb Upper Marker aliquot to room temperature for about 30 min.

2. In an Eppendorf LoBind 0.5 mL tube (provided), aliquot 297 L of the DNF-498 0.25x TE Dilution Buffer.

3. Mix the 165 kb Upper Marker aliquot very slowly by pipetting 1-2 times with a wide-bore genomic pipette tip and a pipettor set to ~5 L volume.

4. Using a regular pipette tip, immediately aliquot 3 L of the mixed 165 kb Upper Marker into the tube containing 297 L of the 0.25x TE Dilution Buffer from step 2. Do not pipette up-and-down or vortex.

5. Using the wide-bore genomic pipette tip only and a pipettor set to a 24 L volume slowly pipette the prepared 165 kb Upper Marker working solution up and down 5 times to mix. This is the 165 kb Upper Marker Working Solution; use within one day of preparation.

6. Using a wide bore tip load 24 L of the 165 kb Upper Marker Working Solution into row A of a 96-well sample plate. Overlay each well with a drop of mineral oil (provided) to prevent evaporation. The prepared 165 kb Upper Marker Working Solution can be used for multiple injections within one day of preparation.

7. The 96-well sample plate containing the 165 kb Upper Marker Working Solution should be placed into Drawer M (third from top) of the Femto Pulse. Ensure that the plate is loaded with well A1 toward the back left on the tray.

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165 kb BAC Ladder Handling and Storage

Prior to first use, the 165 kb BAC Ladder should be aliquoted to minimize the number of freeze/thaw cycles.

1. Equilibrate the 165 kb BAC Ladder to room temperature for about 30 min and mix by gently pipetting up-and-down up to 3 times with wide-bore genomic pipette tips (provided with kit) and a pipettor set to a 15 L volume. Do not vortex or flick the Ladder tube. Only wide-bore genomic pipette tips should be used to mix the 165 kb BAC Ladder solution.

2. Aliquot the 165 kb BAC Ladder solution into 7 aliquots, 5 L each, using the provided wide-bore genomic tips and the provided and the provided Eppendorf LoBind 0.5 mL tubes. Each aliquot is good for 4-times use (1 L per use).

3. Store the 165 kb BAC Ladder aliquots at -20C. Store the in-use vial of the 165 kb BAC Ladder at 2-8C for up to 2 weeks. Avoid freeze-thawing of the 165 kb BAC Ladder more than 4 times (more frequent freeze-thawing may result in degradation of the large size fragments in the 165 kb BAC Ladder).

165 kb Ladder Working Solution Preparation

1. Before use, equilibrate the 165 kb BAC Ladder aliquot to room temperature for about 30 min.

2. In an Eppendorf LoBind 0.5 mL tube (provided), aliquot 9 L of the DNF-498 0.25x TE Dilution Buffer.

3. To the same Eppendorf tube aliquot 90 L of the FP-8001 gDNA Diluent Marker solution. Mix the contents of the tube by vortexing.

4. Mix the 165 kb BAC Ladder aliquot very slowly by pipetting 1-2 times with a wide-bore genomic pipette tip and a pipettor set to ~5 L volume.

5. Using a regular pipette tip, immediately aliquot 1 L of the mixed 165 kb BAC Ladder into the tube containing the 0.25x TE Dilution Buffer and gDNA Diluent Marker from steps 2-3 above. Do not Pipette up-and-down or vortex.

6. Using the wide-bore genomic pipette tip only and a pipettor set to a 20 L volume slowly pipette the prepared 165 kb BAC Ladder working solution up and down 5 times to mix. This is the 165 kb BAC Ladder Working Solution; use within one day of preparation.

7. Load 20 L of the 165 kb BAC Ladder Working Solution into Well 12 of a sample plate row that is to be analyzed.

8. The 165 kb BAC Ladder should be run in parallel with the samples for each experiment. It is not recommended to import a previously run 165 kb BAC Ladder.

NOTE: For samples that do not require predilution or the predilution is less than 160x, the sample matrix is expected to influence sample sizing. Please refer to Appendix: Preparation of 165 kb BAC Ladder Working Solution for instructions on preparing a 165 kb BAC Ladder Working Solution that contains sample matrix.

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Sample Plate Preparation

NOTE: Important BAC fragments Sampling Procedures.

Before sampling, the sample stock BAC fragments must be acclimatized to room temperature for at least 30 minutes.

When mixing BAC samples containing large fragments, slowly pipette up-and-down with wide-bore genomic pipette tips.

Smaller BAC fragments should be run at a lower limit of the concentration range; larger BAC fragments should be run at a higher limit of the concentration range.

Avoid total DNA input sample concentrations above the specified limits. Overloading of DNA sample can result in saturation of the CCD detector and poor results. The peak heights for individual DNA fragments should lie in an optimal range between 1005,000 RFUs.

The sample plate should be analyzed within a day after preparation.

1. If the starting material is higher than the specified concentration, pre-dilute the sample to the specified concentration range with DNF-498 Dilution Buffer 0.25x TE.

2. The sample matrix, i.e. salt concentration in the restriction digestion buffers, can affect the mobility of the DNA fragments and therefore the sample sizing accuracy. Pre-diluting the BAC samples 160-times or more usually nullifies the sample matrix effect on sample sizing.

3. If the sample predilution is less than 160x or not required due to a low initial sample concentration, refer to Appendix A: Preparation of for special instructions to compensate for the matrix effect on the sample migration.

4. Using a clean 96-well sample plate, pipette 18 L of FP-8001 gDNA Diluent Marker Solution (DM) to each well of the 96- well plate to contain a sample. Fill any unused wells within the row of the sample plate with 20 L/well of BF-P25 Blank Solution.

5. Pipette 2 L of each BAC sample into the 18 L of DM in the respective wells of the Sample Plate. Mix the sample wells by pipetting up/down 2-3 times with a wide-bore genomic pipette tip and the pipettor set to ~18 L volume.

6. Load 20 L of the 165 kb BAC Ladder Working Solution into Well 12 of a sample plate row that is to be analyzed (See previous Section).

7. After loading the samples and 165 kb BAC Ladder Working Ladder in each well, check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells. Centrifuge the plate to remove any trapped air bubbles. The presence of trapped air bubbles can lead to injection failures.

8. Run the sample plate immediately once prepared, or cover the sample plate with a cover film, store at 2-8C, and use as soon as possible. Alternatively, to prevent evaporation, place a mineral oil overlay on each sample (20 L/well).

9. To run the samples, place the plate in one of the three sample plate trays (Drawers 4-6 from the top) of the Femto Pulse instrument. Load the experimental method as described in the following sections.

Gel preparation

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Prepare the gel/dye mixture for Femto Pulse System. To ensure the gel/dye mixture is mixed homogeneously without generating bubbles, gently invert the centrifuge tube 5 to 10 times, depending on the volume of the mixture.

NOTE: Centrifuge dye prior to opening the vial to reduce risk of leaking, when possible.

Femto Pulse system volume specifications

# of Samples to be Analyzed1

Volume of Intercalating Dye

Volume of Separation Gel

Volume of 1x Conditioning Solution

12 1.0 L 10 mL 10 mL

24 2.0 L 20 mL 20 mL

36 3.0 L 30 mL 30 mL

48 4.0 L 40 mL 40 mL

96 8.0 L 80 mL 80 mL

1 One sample well per separation is dedicated to the ladder.

Daily Conditioning (Recommended) For optimal array performance when running the FP-1004-0275 165 kb BAC kit, it is recommended to perform an additional daily conditioning of the capillary array for 20 min.

1. From the main screen of the Femto Pulse controller software, select the Operation tab. Under the Capillary Array >

Conditioning field press Add to queue. The Select Conditioning Method form will be displayed, enabling the user to

select the conditioning method from the dropdown menu.

2. Select the 20 min Conditioning method from the dropdown menu. This method performs a 20 min conditioning

solution flush followed by a 3 min Gel fill.

a Ensure there is 50 mL Conditioning Solution loaded on the system.

b Ensure 15 mL PF-50001 FP Large DNA Separation Gel is loaded on the system.

3. Press OK to add the method to the instrument queue (press Cancel to abort adding the method).

4. Press the Play icon to start the sequence loaded into the queue.

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Agilent FP-1004 165 kb BAC assay operating procedure

1. Mix fresh gel and dye according to the volumes in the Gel preparation tables. Refill 1x Conditioning Solution as needed.

2. Place a fresh 1x Inlet Buffer in drawer B on the system, 1.0 mL/well. Replace daily.

2.1. Femto Pulse system; Fill row A of buffer plate

3. Prepare Storage Solution plate. Replace every 2 weeks for optimal results.

3.1. Femto Pulse system; Fill row H of buffer plate with 1.0mL/well, place in drawer B

4. Load 165 kb Upper Marker Working Solution plate (prepared above) in drawer M.

5. Load sample/ladder plate (prepared above) in one of the three sample plate trays (Drawers 4-6 from the top) of the Femto Pulse instrument.

Agilent Femto Pulse software operating procedure

1. Select Row, Group or Tray to run.

2. Enter sample ID and Tray ID (optional).

3. Select Add to Queue, from the dropdown menus select the corresponding method based on your capillary length;

3.1 FP-1004-22 165kb BAC

4. Enter Tray Name, Folder Prefix, and Notes (optional).

5. Select OK to add method to the queue.

6. Select to start the separation.

WARNING Working with Chemicals

The handling of reagents and chemicals might hold health risks.

Refer to product material safety datasheets for further chemical and biological safety information.

Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

Femto Pulse system; Working Ladder Solution well 12, depending on which row is chosen

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165 kb BAC Ladder Result

165 kb BAC Ladder result, using the Femto Pulse System with the FP-1004 165 kb BAC kit. Peaks are annotated by size (bp). Method: FP-1004-22 165kb BAC

Troubleshooting The following table lists several potential assay specific issues which may be encountered when using the FP-1004 165 kb BAC kit and suggested remedies. Contact Agilent local technical support if you have any additional troubleshooting or maintenance questions.

Issue Cause Corrective Action

The 165 kb BAC Upper Marker or Ladder peaks are low, split, or not detected.

1 The 165 kb BAC Upper Marker or Ladder has degraded, or handling instructions have not been followed.

2 The 165 kb BAC Upper Marker or Ladder was vortexed or pipetted with regular pipette tips (not wide-bore).

3 The Femto Pulse capillary array requires a daily 20 min conditioning and Method D flush.

4 The 165 kb BAC Ladder solution is too old.

1 Start with a new aliquot of the 165 kb BAC Upper Marker or Ladder. Prepare and handle the ladder as directed in the manual.

2 Handle the Upper Marker or ladder as directed in the manual.

3 Perform the Method D Flush 0.5 N NaOH Conditioning Gel conditioning method. Perform 20 min conditioning daily.

4 Prepare a fresh 165 kb BAC Ladder solution. Ensure that a new 165 kb BAC Ladder Working Solution is prepared daily.

The peaks of the BAC sample appear spiky or split. 1 Input sample concentration is too

high. 1 Further dilute input sample with DNF-498 Dilution Buffer 0.25x TE and repeat the experiment.

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The peak signal is >>5,000 RFU. 1 Input sample concentration too high. 1 Further dilute input sample concentration with 0.25x TE buffer and repeat the experiment OR Reduce injection time and/or injection voltage and repeat the experiment.

No expected DNA fragment peak(s) observed. Lower Marker observed.

1 Sample concentration is below

detection limit. 2 Sample was not added to a sample

plate, or wrong sample row was selected for analysis.

1 Prepare more concentrated sample

and repeat the experiment. 2 Verify sample was correctly added

or the correct sample row was selected for the analysis.

BAC fragment sizes differ by greater than 15% from expected sizes.

1 Possible sample matrix effect on

sample sizing, particularly if

sample dilution is less than 160x.

2 Input sample concentration is too

high.

1 For important instructions, refer to

the Preparation 165 kb BAC Ladder

working solution.

2 Further dilute input sample with

DNF-498 Dilution Buffer 0.25x TE

and repeat the experiment.

No sample peak or marker peak observed for individual sample.

1 Air trapped at the bottom of sample

plate and/or marker plate well, or

bubbles present in well.

2 Insufficient sample volume. A

minimum of 20 L is required.

3 Capillary is plugged.

1 Check sample/marker plate wells

for trapped air bubbles. Centrifuge

plate.

2 Verify proper volume of solution was

added to sample well and marker

well.

3 Check waste plate for liquid in the

capillary well. If no liquid is

observed, follow the steps outline in

the Appendix Capillary Array

Cleaning of the Femto Pulse User

Manual for unclogging a capillary

array.

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Appendix A: Preparation of 165 kb BAC Ladder Working Solution by Adding Sample Matrix for Optimal Sample Sizing

The presence of salts in BAC samples digested with restriction enzymes can affect the migration of the

fragments relative to the sizing ladder, and thus impact the sizing accuracy of the samples. To account for

sample matrix (salt concentration) effects on the mobility of DNA fragments, it is recommended to dilute the

BAC ladder with the sample matrix.

The ideal sample matrix is the same restriction enzyme buffer that was used to digest the BAC clone samples.

The sample matrix should be added to the ladder at the same final concentration as is present in the prepared

samples

Note: When performing sample pre-dilutions above 160x, it is generally not necessary to add sample matrix to

the 165 kb BAC Ladder working solution, as any sample matrix effects are eliminated by dilution.

165 kb BAC Ladder working solution preparation using the sample matrix

1. Dilute the restriction digestion buffer used for BAC samples to a 1x concentration using nuclease free water. This is the 1x Sample Matrix Solution (1x SMS).

2. Dilute the 1x SMS, 10x using Dilution Buffer 0.25x TE (e.g., add 2 L of the 1x SMS to 18 L of Dilution Buffer 0.25x TE ). This is now the 0.1x Sample Matrix Solution (0.1x SMS).

3. Refer to Table A1 below for example sample dilutions and corresponding volumes of components to be added to make the 165 kb BAC Ladder working solution.

Table A1 Preparation of 165 kb BAC Ladder working solution with sample matrix.

Sample Predilution

FP-8001 DM, L Sample Matrix Solution (SMS), L

DNF-498 Dilution Buffer 0.25x TE, L

165 kb BAC Ladder, L

Total Volume of Ladder Working Solution, L

None 89.0 10.0 (1x SMS) None 1 100

10x 89.0 10.0 (0.1x SMS) None 1 100

20x 90.0 5.0 (0.1x SMS) 4.0 1 100

40x 90.0 2.5 (0.1x SMS) 6.5 1 100

60x 90.0 1.67 (0.1x SMS) 7.33 1 100

80x 90.0 1.25 (0.1x SMS) 7.75 1 100

100x 90.0 1.0 (0.1x SMS) 8.0 1 100

120x 90.0 0.83 (0.1x SMS) 8.17 1 100

140x 90.0 0.71 (0.1x SMS) 8.29 1 100

160x 90.0 0.63 (0.1x SMS) 8.37 1 100

4. If sample pre-dilutions other than those listed in the Table A1 are used, refer to the general formulas below to calculate the volumes of FP-8001 DM solution, SMS and Dilution Buffer 0.25x TE to be added to make the ladder working solution.

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For sample pre-dilutions less than 10x:

a (10/sample pre-dilution) = Volume (L) 1x SMS

b 100 1x SMS Volume (L) 90.0 L (DM solution) 1 L (165 kb BAC Ladder Volume)

= Volume (L) of Dilution Buffer 0.25x TE

c Combine the components using the calculated volumes (L) from A and B above:

i L 1x SMS (from calculation A)

ii 90.0 L DM solution

iii L Dilution Buffer 0.25x TE (from calculation B)

iv 1 L 165 kb BAC Ladder

d The total volume of the ladder working solution should equal 100 L.

For sample pre-dilutions greater than 10x:

a (100/sample pre-dilution) = Volume (L) 0.1x SMS

b 100 0.1x SMS Volume (L) 90.0 L (DM solution) 1 L (165 kb BAC Ladder

Volume) = Volume (L) of Dilution Buffer 0.25x TE

c Combine the components using the calculated volumes (L) from A and B above:

i L 0.1x SMS (from calculation A)

ii 90.0 L DM solution

iii L Dilution Buffer 0.25x TE (from calculation B)

iv 1 L 165 kb BAC Ladder

d The total volume of the ladder working solution should equal 100 L.

5. Following preparation of the 165 kb BAC Ladder working solution with sample matrix, load 20 L of the prepared 165 kb BAC Ladder working solution into well 12 of a sample plate row that is to be analyzed.

6. The 165 kb BAC Ladder should be run in parallel with the samples for each experiment. It is not recommended to import a previously run sizing ladder.

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Appendix B: DNA fragment analysis without using the Upper Marker in the sample wells

The FP-8004 165 kb Upper Marker (UM) can co-migrate with DNA fragments above 100 kb interfering with fragment sizing. Instructions for preparation of the Upper Marker row when the 165 kb UM is NOT desired in the samples are outlined below.

Femto Pulse Upper Marker Plate Preparation

1. Before use, equilibrate the 165kb Upper Marker aliquot to room temperature for about 30 min.

2. In an Eppendorf LoBind 0.5 mL tube (provided), aliquot 99 L of the DNF-498 0.25X TE Dilution Buffer.

3. Mix the 165 kb Upper Marker aliquot very slowly by pipetting 1-2 times with a wide-bore genomic pipette tip and a pipettor set to ~5 L volume.

4. Using a regular pipette tip, immediately aliquot 1 L of the mixed 165 kb Upper Marker into the tube containing 99 L of the 0.25X TE Dilution Buffer from step 2. DO NOT pipette up-and-down or vortex.

5. Using the wide-bore genomic pipette tip only and a pipettor set to a 24 L volume slowly pipette the prepared 165 kb Upper Marker working solution up and down 5 times to mix. This is the 165kb Upper Marker Working Solution; use within one day of preparation.

6. Using a wide bore tip load 24 L of the 165 kb Upper Marker Working Solution into ONLY well 12 of row A (the Upper Marker row) of a 96-well sample plate.

7. Using a regular pipette tip pipette 24 L of the DNF-498 0.25X TE Dilution buffer in wells 1-11 of row A (the Upper Marker Row).

8. Overlay each well with a drop of mineral oil (provided) to prevent evaporation. The prepared 165 kb Upper Marker Working Solution can be used for multiple injections within one day of preparation.

9. The 96-well sample plate containing the 165 kb Upper Marker Working Solution should be placed into Drawer M (third from top) of the Femto Pulse. Ensure that the plate is loaded with well A1 toward the back left on the tray.

ProSize Data Analysis Settings

1. In the ProSize data analysis software select the Marker Analysis settings (Figure B1).

2. Click on the drop-down menu as shown in Figure B1 and select Use Lower Marker Only.

3. Click Apply to All.

4. The Upper Marker will be displayed as a 165 kb peak in the ladder, and there will be no Upper Marker designation in the sample wells.

5. Without Upper Marker, sizing accuracy can exceed the specification of 15%.

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Figure B1. ProSize data analysis software window for selecting the Lower Marker Only analysis option.

From the dropdown menu select Use Lower Marker Only.

Click Apply to All

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Appendix C: Method D Flush

Occasionally when performing separations of high molecular weight (HMW) DNA > 100kb, loss of HMW DNA peak shape or

peak signal can occur. In such cases, it is recommended to perform an additional cleaning of the capillary array with 0.5 N

NaOH solution, conditioning solution, and gel using the Method D Flush to restore separation performance. Instructions for

performing this protocol are outlined below.

WARNING

0.5 N NaOH is corrosive

Use extreme caution when handling, as exposure can cause severe eye and skin burns. Avoid

contact with eyes, skin, or clothing. Wear eye protection and impervious gloves. Clearly label

containers to avoid accidental exposure.

Refer to the product material safety datasheets for all warnings and precautions before

proceeding.

1. From the main screen of the Femto Pulse controller software, select the Operation tab. Under the Capillary Array > Conditioning field click Add to queue. The Select Conditioning Method form will be displayed, enabling the user to select the conditioning method from the dropdown menu.

2. Select Method D Flush 0.5 N NaOH Conditioning Gel from the method dropdown menu. This method will perform a 20 min 0.5 N NaOH solution flush from the Gel2 fluid line, a 20 min conditioning solution flush from the Conditioning line, and a 3 min gel flush from the Gel1 line (figure below).

3. Click OK to add the method to the instrument queue (click Cancel to abort adding the method).

4. Open the Femto Pulse system side compartment and replace the Gel2 bottle with a bottle containing a minimum of 25 mL of 0.5N NaOH solution.

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5. Ensure there is a minimum of 25 mL of 1x Conditioning Solution in the Conditioning bottle, and a minimum of 10 mL of FP Large DNA Separation Gel in the Gel1 bottle.

6. Close the door to the system side compartment and click the Play icon to start the selected capillary conditioning

method.

7. Once the capillary conditioning method is complete, open the waste drawer and remove the 96-deepwell 1mL plate. Empty the waste plate contents in the proper waste disposal area and return the empty plate to the waste drawer.

8. The Femto Pulse system is now ready to run additional samples or can be stored until next use.

For Research Use Only Not for use in Diagnostic Procedures.

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