Biotage PhyPrep Bedienungsanleitung

Biotage®PhyPrep

User Manual

Biotage®PhyPrep User Manual | © Biotage 2021

Biotage® PhyPrep

Biotage®PhyPrep

User Manual

CONTENTS

4System Overview

 Quickstart Guide

 Hardware & Connections

 Instrument Safety

 Sample Filter Reservoir

 Biotage®PhyPrep Plasmid Kit Endotoxin-Free

 Instrument Deck

9Before You Begin

 Buffer Preparation

 Recommended Culture Conditions for Optimal Results

10 Sample Preparation Guide

 Resuspend Cells

 Lyse Cells

 Precipitate

 Filter

12 Maxiprep Sample Preparation

 Maxiprep  Sample Method

 Maxiprep  Sample Deck Layout

 Maxiprep Samples Method

 Maxiprep  Samples Deck Layout

 Maxiprep Samples Method

 Maxiprep  Samples Deck Layout

 Maxiprep Samples Method

 Maxiprep  Samples Deck Layout

20 Megaprep Sample Preparation

 Megaprep  Sample Method

 Megaprep  Sample Deck Layout

 Megaprep Samples Method

 Megaprep  Samples Deck Layout

 Megaprep Samples Method

 Megaprep  Samples Deck Layout

 Megaprep Samples Method

 Megaprep  Samples Deck Layout

28 Gigaprep Sample Preparation

 Gigaprep  Sample Method

 Gigaprep  Sample Deck Layout

 Gigaprep Samples Method

 Gigaprep  Samples Deck Layout

 Gigaprep Samples Method

 Gigaprep  Samples Deck Layout

 Gigaprep Samples Method

 Gigaprep  Samples Deck Layout

36 Software Overview

37 Recommended Alcohol Precipitation protocol

38 Maintenance

 Endotoxin Levels

 Using the endotoxin measuring instrument

 Wash Buffer Reservoir

 Carboys Set Up and Care

 Carboys Contents Disposal

40 Troubleshooting

 Low DNA yield

 Low DNA quality

42 General Information

 Consumables and Accessories



Biotage® PhyPrep

Biotage®PhyPrep User Manual | © Biotage 2021

System Overview

The Biotage®PhyPrep system is the only all-in-one instrument for automated endotoxin-free plasmid purification in maxi, mega, and

giga scales. This system offers a variety of user friendly protocols for endotoxin-free plasmid purification.

It is developed and supported by scientists and engineers to provide consistent high-quality endotoxin-free plasmids for transient

transfection.

System Overview

Prep Scale

Number

of Sample

Preps

Run Time

(Hour/Minutes)

Culture Volume

Per Sample

Pellet

Wet Weight

Per Sample

Optimal Pellet

Wet weight

Plasmid

Mass

Elution

Volume

Maxiprep

142 m

150–250 mL 3-5 g 3 g Up to

1 mg 5 mL

242 m

31 h 10 m

41 h 10 m

Megaprep

11 h 16 m

350–500 mL 6-8 g 7 g Up to

5 mg 18 mL

21 h 16 m

32 h 22 m

42 h 22 m

Gigaprep

12 h 31 m

60 0 –750 mL 14-16 g 15 g Up to

10 mg 28 mL

22 h 34 m

34 h 57 m

44 h 58 m

Quickstart Guide

Choose

your scale

Determine the culture volume and culture media needed for generating pellet wet weight of

E-coli

cells for your prep scale

It is recommended to use TB or other high growth media for optimal yield.

Grow culture Grow overnight cultures and harvest the cell pellets by centrifugation.

Select

Sample Size

Choose the number of samples to purify. Power up the instrument and select your prep scale and

number of your samples in the PhyPrep software.

Set Up Deck Set up the deck with consumables as described in the protocols.

Prepare

Culture

Resuspend, lyse, and precipitate the sample(s) using the color-coded buffer system.

Pour the neutralized lysate into the Sample Filter Reservoirs on the instrument.

Run Method Start the method in the PhyPrep software to start the run.

Collect

Plasmids

When the run is complete, collect the elution tubes from the elution stand. Use immediately for

downstream assays or refrigerate or freeze for later use.





Hardware & Connections

Parts

Instrument Side Panel

1. Power connector

2. Power switch

3. Vacuum power connector

4. Ethernet port

5. PC power connector

6. Vacuum port and red tubing

7. Waste port and blue tubing

Emergency Stop

Open the door for

emergency stop.

Panel PC

Leakage Tray

Waste

Carboy

Vacuum

pump

Vacuum

Carboy

Vacuum Waste

Vacuum

Carboy

Waste

Carboy

Sample

Filter

Reservoir

Elution Tube

Stand

Wash Buffer

Reservoir

Vacuum Växel

Column

Transfer

Tip

Elution

Tube

PhyPrep

Tube

Biotage®PhyPrep User Manual | © Biotage 2021

Instrument Safety

Open Section

Sample Filter Reservoir

Assembly

Assemble the Sample Filter Reservoirs with the correct orientation

before placing them on the deck. The reservoir consists of a filter

top and a reservoir bottom. The filter lid has an open section and

the reservoir has a large edge and a small edge for orientation.

To assemble the Sample Filter Reservoir, place the open section of

the filter top over the small edge of the reservoir bottom.

Placement on deck

Place the Sample Filter Reservoir on the deck with the filter

section closest to the front of the instrument.

Instrument Safety

Emergency Stop

The PhyPrep door works as an emergency stop. If the door is

opened at any time during system operation, the instrument

will stop operating immediately, and the current method will be

aborted. It is not possible to resume the stopped method again

after PhyPrep door is opened.

After an emergency stop, if the pipetting head has transfer tips

or Växel columns attached, eject these by using the “Initialize”

button in the software. There should not be any columns attached

to the pipetting head before starting a new run.

Important note

Never try to take off attached transfer tips or Växel columns

manually as you may harm your hand.

The STOP Button

If the STOP button is pressed during a method run, you are asked

to confirm that the method should be stopped. If you confirm, the

system will stop, and the current method will be aborted. It is not

possible to resume the method after pressing STOP.

Vacuum Pump Voltage

Ensure the voltage on the vacuum pump is set correctly.

Elution Tube Stand

For each sample being purified, be sure

to place a  mL pyrogen-free elution

tube in the bottom level of the elution

stand.

Instrument Cleaning

Do not spray alcohol direclty onto the instrument robotics parts.

Doing so will lead to damages to your PhyPrep.

Ensure paper towels and or other materials are not placed in the

vacuum station.

Filter Reservoir

Filter

Reservoir

Open section

Small edge

Large edge

Open section

Small edgeLarge edge

 corresponds to

- volts

 corresponds to

- volts

Elution

stand

Elution

collection

tubes



Biotage® PhyPrep Plasmid Kit Endotoxin-Free

Biotage®PhyPrep Plasmid Kit Endotoxin-Free

Kit Contents

Maxiprep kit DPM-16-05-72-KIT

710-0001 900-0078 900-0081 900-0084 900-0091 900-0093 900-0087 900-0089 700-0030 DPM -16- 05-

72-RNA 417458SP DPM-00-

25-00

16 x 16 x 16 x 16 x 1 x 16 x 1 x 1 x 16 x 16 x 50 x 50 x

Växel

Maxiprep

Column

Resuspension

Buffer

Lysis

Buffer

Precipitation

Buffer

Wash

Buffer

Elution

Buffer

Endotoxin

Removal

Buffer

Equilibration

Buffer

Sample Filter

Reservoir

RNase A

Tubes

PhyPrep

Tube

20 mL

Transfer Tip

Megaprep kit DPM-16-10-72-KIT

710-0002 900-0079 900-0082 900-0085 900-0092 900-0094 900-0087 900-0089 700-0030 DPM -16-10 -

72-RNA 417458SP DPM-00-

25-00

16 x 16 x 16 x 16 x 1 x 16 x 1 x 1 x 16 x 16 x 100 x 50 x

Växel

Megaprep

Column

Resuspension

Buffer

Lysis

Buffer

Precipitation

Buffer

Wash

Buffer

Elution

Buffer

Endotoxin

Removal

Buffer

Equilibration

Buffer

Sample Filter

Reservoir

RNase A

Tubes

PhyPrep

Tube

20 mL

Transfer Tip

Gigaprep kit DPM-08-20-72-KIT

710-0003 900-0080 900-0083 900-0086 900-0092 900-0094 900-0088 900-0090 700-0030 DPM-08-20-

72-RNA 417458SP DPM-00-

25-00

8 x 8 x 8 x 8 x 1 x 8 x 1 x 1 x 8 x 8 x 50 x 25 x

Växel

Gigaprep

Column

Resuspension

Buffer

Lysis

Buffer

Precipitation

Buffer

Wash

Buffer

Elution

Buffer

Endotoxin

Removal

Buffer

Equilibration

Buffer

Sample Filter

Reservoir

RNase A

Tubes

PhyPrep

Tube

20 mL

Transfer Tip

Biotage®PhyPrep User Manual | © Biotage 2021



Instrument Deck

The Biotage PhyPrep has specific deck layouts for all of the

pre-programmed methods.

Section 1: Transfer tips

Tips are placed in the upper left corner.

Section 2: Vacuum and waste station

Section 3: Wash Buffer Reservoir

The Wash Buffer Reservoir is located in the upper

right corner. It should be removed from the deck prior

to filling it with wash buffer to minimize the risk of

spilling. Empty remaining volume after each run and

clean with % alcohol.

Section 4: PhyPrep Tubes and Växel columns

This section contains space for  Växel columns

and  PhyPrep Tubes.

Section 5: Supplemental buffer section

The supplemental buffer section contains  rows of

PhyPrep Tube spots. Each row contains space for 

tubes.

Figure 1. The PhyPrep deck layout 1) Transfer tubes 2) Vacuum station 3) Wash Buffer Reservoir 4) Biotage Växel columns and PhyPrep Tubes

5) Supplemental buffers 6) Biotage Växel columns and PhyPrep Tubes 7) Elution Tube Stand 8) Sample Filter Reservoirs

Section 6: PhyPrep Tubes and Växel columns

This section contains space for  Biotage Växel

columns and  PhyPrep Tubes.

Section 7: Elution Tube Stand

The Elution Tube Stand is located near

the front center. For each sample being

purified, a  mL pyrogen-free elution

tube must be placed in the bottom level

of the elution stand. Before collecting

the purified sample, carefully remove

the Växel columns from the Elution

Tube Stand. There may be a residual

drop attached to the bottom of each tip.

Lightly tap the drop onto the elution tube

you would like to collect.

Section 8: Sample Filter Reservoirs

The Sample Filter Reservoirs are located in the

frontmost portion of the deck. For each sample

being purified, one sample reservoir should be

used. Place the Filter Reservoirs in the correct

orientation with the filter closest to the front of

the deck.

Elution

collection

tube

Wash Buffer

Reservoir

Sample Filter

Reservoir

Transfer

Tip

PhyPrep

Tube

Elution

tube

stand

PhyPrep

Tube

PhyPrep

Tube



Before You Begin

Buffer Preparation

Add the provided lyophilized RNase A to

Resuspension Buffer before use. Once RNase A is

added to Resuspension Buffer, the solution should

be stored at –˚C if not immediately used.

Check Lysis Buffer for precipitation due to low

storage temperature. In the case of precipitation,

dissolve by warming bottle(s) in water bath at

–˚C for – minutes and mix by inversion.

Store these buffers at ˚C overnight before use

for optimal yield:

Resuspension Buffer

Precipitation Buffer

Endotoxin Removal Buffer

Equilibration Buffer

To maintain the sterility of the equilibration buffer,

endotoxin removal buffer, and wash buffer when

aliquoting, use the fume hood and a flame. With the flame

and fume hood on, pass the buffer container lid and neck

through the flame and open the bottle while hovering

near the flame.

When pouring out the specified amount of buffer,

pour the buffer from the bulk bottle into the

specified container for the deck while hovering

near the flame. After aliquoting, flame through the

cap and opening of the bottle before closing.

Preparing Resuspension Buffer

You can add the RNase A to Resuspension Buffer when you

receive your kit. Once RNase A has been added to

Resuspension Buffer , store at –˚C.

Recommended Culture Conditions

for Optimal Results

The Biotage PhyPrep’s robust instrumentation and chemistry

allows for dense sample processing without the worry for

clogging or contamination. Because of the instrument’s

flexibility and scale, Biotage recommends growing denser

cultures compared to traditional manual preps.

Please follow the outlined recommendations for optimizing

culture growth:

Growth Medias

The use of rich medias has the highest impact on cell growth

density.

We recommend the following:

Terrific Broth (TB)

Thomson Plasmid+®

Agencourt Ale

Ratio of Media to Flask Size

Use a : ratio of media to flask size. The volume of media

should not exceed % of the flask volume. This is to allow

proper aeration of the culture during incubation and maximize

cell division.

Incubator Temperature and Shaking Frequency

We recommend incubating at °C and  RPM. Larger

cultures require faster shaking speeds to properly aerate the

whole volume of cells. Certain plasmids (for example pUC)

will increase in copy number when grown at slightly higher

temperatures.

Starter and Overnight Culture Times

A smaller culture grown for – hours to inoculate the overnight

culture at a dilution of : is recommended. In rich media,

cultures may grow slightly longer for about  hours overnight.

Growth Flask and Air Permeable Seal

Cell density in cultures can be further optimized using Thomson

flasks and seals.

Thomson Optimal Growth Flasks

Thomson AirOtop™Enhanced Seals

Harvest Cells



 g pellet wet weight is needed for Maxiprep, achieved

by growing  mL of an overnight bacterial culture.



 g pellet wet weight is needed for Megaprep, achieved

by growing  mL of an overnight bacterial culture.



 g pellet wet weight is needed for Gigaprep, achieved

by growing  mL of an overnight bacterial culture.

Centrifuge at ≥  x g for  minutes. Discard supernatant.

The PhyPrep deck can be set up while waiting for cells to

pellet. See appropriate deck layout per desired method

for further instructions.

Sample Preparation Guide

All steps are carried out at room temperature. Please review the section “Before You Begin” on page 9 prior to

proceeding with sample preparation.

. Resuspend Cells

Add contents of RNase A tube to the RESUSPENSION BUFFER. Uncap the centrifuge bottle and add the entire contents

of RESUSPENSION BUFFER. To aid in pellet resuspension, agitation beads are included in RESUSPENSION BUFFER.

Recap the centrifuge bottle and shake vigorously until the bacterial pellet is completely resuspended.

Important reminder

Use 1 Resuspension Buffer bottle per sample. Verify that RNase A is added to Resuspension Buffer.

Important notes

Incomplete resuspension can result in poor recovery of plasmid DNA.

. Lyse Cells

Uncap the centrifuge bottle and add the entire contents of LYSIS BUFFER. Recap the centrifuge bottle and mix by

inverting gently – times. To aid in the lysis step, a blue indicator dye is present in LYSIS BUFFER. Ensure the

solution turns completely blue. If nonblue portions are observed after  inversions, continue mixing gently.

Let the lysis step occur for at least  minutes. Do not let the lysis step exceed  minutes.

Important reminder

Use 1 Lysis Buffer bottle per sample. Verify that Lysis Buffer has not precipitated.

Important notes

Do not shake or vortex during this step. Harsh mixing will shear genomic DNA and may contaminate the

final recovered plasmid DNA.

Do not allow lysis to proceed longer than 5 minutes. Prolonged alkaline lysis may permanently denature

the supercoiled plasmid DNA and may render it unsuitable for use in downstream applications.

If the buffer contents have precipitated due to low temperatures, warm bottle to 30–37°C for 10–20 min

and shake to dissolve potential precipitation.

. Precipitate

Uncap the centrifuge bottle and add the entire contents of PRECIPITATION BUFFER. Recap the centrifuge bottle and mix by

inverting - times. A white precipitate containing cell debris, proteins, lipids, SDS, and chromosomal DNA will form.

Upon complete neutralization, the solution will turn from blue to clear. If blue portions are observed after  inversions,

continue mixing gently.

Important reminder Use 1 Preciptation Buffer bottle per sample.

Important notes

Mixing during this step should be gentle but thorough. Although it is important to completely mix the

solution to ensure complete precipitation of cellular debris, do not shake or vortex during this step. Harsh

mixing can cause break up of precipitated debris and may contaminate the final recovered plasmid DNA.

For increased yield, use cold Preciptation Buffer, Equilibration Buffer, Endotoxin Removal Buffer, and

Resuspension Buffer that have been stored at 4°C.

. Filter

Place Sample Filter Reservoir on the PhyPrep deck. Then slowly pour entire contents of the centrifuge bottle into the middle

of the Sample Filter Reservoir to allow the supernatant to go through the filter first. This will prevent the precipitated

particulates clogging the filter.

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