AutoPlasmid MMG User Manual v2 - PhyNexus · AutoPlasmid MMG Kits 3 AutoPlasmid MMG Instrument Deck...

Introduction 1 AutoPlasmid MMG Kits 3 AutoPlasmid MMG Instrument Deck 4-5 Deck Layout Overview 6-7 Before Starting 8 Waste Station Tubing 9 Maxiprep 10-19 Sample preparation 10 1 Sample: protocol 12 1 Sample: deck layout 13 2 Samples: protocol 14 2 Samples: deck layout 15 3 Samples: protocol 16 3 Samples: deck layout 17 4 Samples: protocol 18 4 Samples: deck layout 19 Megaprep 20-29 Sample preparation 20 1 Sample: protocol 22 1 Sample: deck layout 23 2 Samples: protocol 24 2 Samples: deck layout 25 3 Samples: protocol 26 3 Samples: deck layout 27 4 Samples: protocol 28 4 Samples: deck layout 29 Gigaprep 30-35 Sample preparation 30 1 Sample: protocol 32 1 Sample: deck layout 33 2 Samples: protocol 34 2 Samples: deck layout 35 Instrument Software Overview 36-40 Troubleshooting 41-44 Catalog Part Numbers 45

Introduction

AutoPlasmid MMG User Manual v2.10 Page 1

Introduction

Thank you for choosing the AutoPlasmid MMG System from PhyNexus, the only all-in-one instrument for fully automated Maxiprep, Megaprep, and Gigaprep plasmid purifications. This system offers a variety of user friendly protocols for almost any plasmid purification specifications.

The PhyNexus Approach to Maxi, Mega and Giga Plasmid Preps The AutoPlasmid MMG - One instrument to perform whatever scale is needed.

Introduction


The AutoPlasmid MMG requires set-up just one time to run up to 4 maxipreps, 4 megapreps, or 2 gigapreps.

Prep Scale Number of Preps

Run Time (min)

Culture Volume per Prep

(mL)

Maximum Plasmid Mass

(mg)

Typical Plasmid Mass (mg)

Max

ipre

p 1 135

150 - 500 1.5 0.75 2 135 3 220

4 220

Meg

apre

p

1 165

500 - 1500 5 3.5 2 165

3 310

4 310

Gig

apre

p

1 165

1000 - 3000 10 7

2 310

Introduction


AutoPlasmid MMG Kits

Maxiprep Kits (16 samples) Part Number: DPM 16-03-70 Description QTY

Tips

PhyTip Columns (3 mL resin) 16 x

Buf

fers

Resuspension Buffer (48 mL in 60 mL bottles) + resuspension beads (5 mL or 7 g) 16 x Lysis Buffer (57 mL in 90 mL bottles) 16 x Precipitation Buffer (68 mL in 90 mL bottles) 16 x Wash Buffer (B) (130mL in 500 mL bottle; user adds 300 mL EtOH for a total of 430 mL) 1 x Wash Buffer (1184 mL in 1 gallon bottle; user adds 2200mL EtOH for a total of 3384 mL) 1 x Elution Buffer (20 mL) 16 x

Tube

s Elution tubes (5 mL snap cap) 16 x Sample filter plates 16 x RNase A tubes (20 mg) 16 x

Megaprep Kits (16 samples) Part Number: DPM 16-06-70 Description QTY

Tips


Buf

fers


Tube


Gigaprep Kits (8 samples) Part Number: DPM 08-06-70 Description QTY

Tips


Buf

fers


Tube


Introduction


AutoPlasmid MMG Instrument Deck

Introduction


Deck Layout Overview


Deck Layout: Overview

The AutoPlasmid MMG has specific deck layouts for all of the pre-programmed methods. The following pages detail the general layout of the MMG deck. The numbers below correspond to the specific deck sections seen on page 7.

Deck Layout Positions: 1. PhyTip Columns and 20 mL transfer tips

Tips are placed in the upper left corner. There are four types of tips that can be used on the AutoPlasmid MMG system: Maxiprep PhyTip Columns, Megaprep PhyTip Columns, Gigaprep PhyTip Columns, and 20 mL Transfer Tips.

2. Drying and waste stations The drying and waste stations are located in the top center. The waste station is located below the drying station. Tips should never be placed in these stations prior to the start of a method.

3. Wash buffer reservoir The Wash Buffer reservoir is located in the upper right corner. The reservoir should be

removed from the deck prior to filling it with Wash Buffer. This minimizes the risk of spilling.

4. 50 mL conical tube section 50 mL conical tubes are placed in multiple sections. Section 4 contains 8 rows of zig-zagging conical tube spots. Each row contains space for two conical tubes.

5. Supplemental buffer section The supplemental buffer section contains 4 rows of zig-zagging conical tube spots. Each

row contains space for two tubes. 5-A and 5-B are used to place Elution Buffer tubes. 5-C and 5-D are used to place 50 mL conical tubes containing 100% Ethanol. 5-E, 5-F, 5-G and 5-H are used to place 50 mL conical tubes containing Wash Buffer B.

6. 50 mL conical tube section Section 6 contains 6 rows of zig-zagging conical tube spots. Each row containing space for two conical tubes.

7. Elution stand The elution stand is located near the front center. For each sample being purified, a 5 mL elution collection tube most be uncapped and placed in the bottom level of the elution stand. The purified eluent for sample 1 is collected in 7-A, sample 2 in 7-B, sample 3 in 7-C, and sample 4 in 7-D. Before collecting the purified sample, carefully remove the PhyTip columns from the Elution Stand. There may be a residual drop attached to the bottom of each tip.

8. Sample processing plates The sample processing plates are located in the bottom portion of the deck. For each sample being purified, one sample processing plate should be used. Before placing the plate on the deck, make sure to remove the top cover. Also, make sure the plate is in the correct orientation with the filter closest to the front of the deck.

Deck Layout Overview



Before Starting

Buffer Preparation: • Add the provided lyophilized RNase A to BUFFER RB before use. Once RNase A is added

to BUFFER RB, the solution should be stored at 2-8˚C if not immediately used. • Add 100% ethanol to BUFFER WB and BUFFER WB (b) before use. See bottle label for

volumes to add. • Check BUFFER LB for precipitation due to low storage temperature. In the case of

precipitation, dissolve by warming bottle(s) in water bath at 30-37˚C for 10-20 minutes and mix by inversion.

Instrument and Consumable Preparation: • Both 4L and 20L vacuum and waste carboys are available for the MMG instrument. It is

essential to empty the carboys once they reach 80% capacity.

Number of samples completed to reach 80% fill capacity

Prep Type 20 L Waste Carboy 20 L Vacuum Carboy 4 L Vacuum Carboy

Maxiprep 160 95 20 Megaprep 110 45 10 Gigaprep 55 20 5

• Empty remaining volume from WB RESERVOIR if using instrument after a previous run.

• Add deionized H2O to PhyTip Columns until resin is fully saturated. Use a Kimwipe to remove any resin from the top of the PhyTip Column orifice.

Time Saving Measures Although the AutoPlasmid MMG is designed for an easy, fast set up, there are certain preparatory measures you can take to maximize efficiency during the day. Preparing BUFFER RB:

You can add RNase A to BUFFER RB when you receive your kit. Once RNase A has been added to BUFFER RB, store at 2-8˚C.

Pre-aliquoting BUFFER WB (b):

Maxiprep: For every sample purified, 25 mL of WB (b) needs to be aliquot into a 50 mL conical tube. When you receive your kit, bring WB (b) to its working volume by adding 300 mL of 100% ethanol. After, add 25 mL of WB (b) + ethanol to 16 x 50 mL conical tubes. Cap and label these tubes and store at room temperature. Megaprep/Gigaprep: For every sample purified, 50 mL of WB (b) needs to be aliquot into a 50 mL conical tube. When you receive your kit, bring WB (b) to its working volume by adding 300 mL of 100% ethanol. After, add 50 mL of WB (b) + ethanol to 16 x 50 mL conical tubes. Cap and label these tubes and store at room temperature.


Waste Station Tubing

Protocols: Maxiprep


Maxiprep: Sample Preparation All steps are carried out at room temperature. Please review the “Before Starting” section on page 8 prior to proceeding with sample preparation. 1. Harvest Cells

• Pellet 150 – 500 mL of an overnight bacterial culture by centrifugation at ≥ 3500 x g for 30 minutes. Discard supernatant.

• The MMG work deck can be set up while waiting for cells to pellet. See appropriate deck layout per desired method for further instructions.

2. Resuspend Cells • Uncap the centrifuge bottle and add the entire contents of BUFFER RB. To aid in pellet

resuspension, agitation beads are included in BUFFER RB. Recap the centrifuge bottle and shake vigorously until the bacterial pellet is completely resuspended.

Important reminders - Use 1 BUFFER RB bottle per sample. Verify that RNase A is added to BUFFER RB.

Important notes - Incomplete resuspension can result in poor recovery of plasmid DNA. 3. Lyse Cells

• Uncap the centrifuge bottle and add the entire contents of BUFFER LB. Recap the centrifuge bottle and mix by inverting gently 20 - 30 times. To aid in the lysis step, a blue indicator dye is present in BUFFER LB. Ensure the solution turns completely blue. If nonblue portions are observed after 30 inversions, continue mixing gently. Let the lysis step occur for at least 3 minutes. Do not let the lysis step exceed 5 minutes. Important reminders - Use 1 BUFFER LB bottle per sample. Verify that BUFFER LB 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, please incubate until components go into solution. A water bath of hot tap water should suffice.

4. Precipitate

• Uncap the centrifuge bottle and add the entire contents of BUFFER PB. Recap the centrifuge bottle and mix by inverting 20-30 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 30 inversions, continue mixing gently. Important reminders - Use 1 BUFFER PB 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 Buffer PB that has been stored at 4°C.

5. Filter

• Pour entire contents of the centrifuge bottle into the middle of the SAMPLE FILTRATION PLATE.

Protocols: Maxiprep


Maxiprep kit sample preparation guide

To view a video of this procedure, go to www.phynexus.com/mmg-training

http://www.phynexus.com/mmg-training

Protocols: Maxiprep 1 sample


Maxiprep: 1 sample method This protocol is designed for the preparation of up to 1.5 mg high-copy plasmid DNA using the AutoPlasmid MMG Maxiprep Kit. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.

Culture volume range: 150 mL— 500 mL Culture volume recommended: 250 mL

Important notes: Ensure correct volume of Wash Buffer is added to Reservoir in Section 3. Ensure correct volumes of Wash Buffer B and Ethanol are added to 50 mL conical tubes in Section 5. Ensure resin in PhyTip columns are wetted with deionized water.

Deck Layouts: Maxiprep 1 sample


Maxiprep: 1 sample method

Protocols: Maxiprep 2 samples


Maxiprep: 2 samples method This protocol is designed for the preparation of up to 1.5 mg high-copy plasmid DNA using the AutoPlasmid MMG Maxiprep Kit. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.



Deck Layouts: Maxiprep 2 samples


Maxiprep: 2 samples method



Maxiprep: 3 samples method This protocol is designed for the preparation of up to 1.5 mg high-copy plasmid DNA using the AutoPlasmid MMG Maxiprep. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.


Important notes: Ensure correct volume of Wash Buffer is added to Reservoir Section 3. Ensure correct volumes of Wash Buffer B and Ethanol are added to 50 mL conical tubes in Section 5. Ensure resin in PhyTip columns are wetted with deionized water.



Maxiprep: 4 samples method This protocol is designed for the preparation of up to 1.5 mg high-copy plasmid DNA using the AutoPlasmid MMG Maxiprep kit. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.

Culture volume range: 150 mL— 500 mL Culture volume recommended: 250 mL Important notes: Ensure correct volume of Wash Buffer is added to Reservoir Section 3. Ensure correct volumes of Wash Buffer B and Ethanol are added to 50 mL conical tubes in Section 5. Ensure resin in PhyTip columns are wetted with deionized water.

Protocols: Maxiprep


Megaprep: Sample preparation All steps are carried out at room temperature. Please review the “Before Starting” section on page 8 prior to proceeding with Sample preparation. 1. Harvest Cells

• Pellet 500 – 1500 mL of an overnight bacterial culture by centrifugation at ≥ 3500 x g for 30 minutes. Discard supernatant.

• The MMG work deck can be set up while waiting for cells to pellet. See appropriate deck layout per desired method for further instructions.

2. Resuspend Cells • Uncap the centrifuge bottle and add the entire contents of BUFFER RB. To aid in pellet

resuspension, agitation beads are included in BUFFER RB. Recap the centrifuge bottle and shake vigorously until the bacterial pellet is completely resuspended.

Important reminders - Use 1 BUFFER RB bottle per sample. Verify that RNase A is added to BUFFER RB.


• Uncap the centrifuge bottle and add the entire contents of BUFFER LB. Recap the centrifuge bottle and mix by inverting gently 20-30 times. To aid in lysis step, a blue indicator dye is present in BUFFER LB. Ensure the solution turns completely blue. If nonblue portions are observed after 30 inversions, continue mixing gently. Let the lysis step occur for at least 3 minutes. Do not let lysis step exceed 5 minutes. Important reminders - Use 1 BUFFER LB bottle per sample. Verify that BUFFER LB has not

precipitated. Important notes


4. Precipitate

• Uncap the centrifuge bottle and add the entire contents of BUFFER PB. Recap the centrifuge bottle and mix by inverting 20-30 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 30 inversions, continue mixing gently. Important reminders - Use 1 BUFFER PB bottle per sample. Important notes


5. Filter • Pour contents of centrifuge bottle into the middle of the SAMPLE FILTRATION PLATE.

Important notes - Care should be taken to avoid spilling and overflow. Do not add entire contents of centrifuge bottle into the SAMPLE FILTRATION PLATE if filtration occurs slowly. Wait until volume in SAMPLE FILTRATION PLATE has receded before adding the full contents.

Protocols: Maxiprep


Megaprep kit sample preparation guide



Protocols: Megaprep 1 Sample


Megaprep: 1 sample method This protocol is designed for the preparation of up to 5 mg high-copy plasmid DNA using the AutoPlasmid MMG Megaprep. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.

Culture volume range: 500 mL— 1.5 L Culture volume recommended: 1 L


Deck Layouts: Megaprep 1 Sample


Megaprep: 1 sample method

Protocols: Megaprep 2 Samples


Megaprep: 2 samples method This protocol is designed for the preparation of up to 5 mg high-copy plasmid DNA using the AutoPlasmid MMG Megaprep. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.



Deck Layouts: Megaprep 2 Samples


Megaprep: 2 samples method




Culture volume range: 500 mL— 1.5 L Culture volume recommended: 1 L Important notes: Ensure correct volume of Wash Buffer is added to Reservoir in Section 3. Ensure correct volumes of Wash Buffer B and Ethanol are added to 50 mL conical tubes in Section 5. Ensure resin in PhyTip columns are wetted with deionized water.





Important notes: Ensure correct volume of Wash Buffer is added to Reservoir Section 3. Ensure correct volumes of Wash Buffer B and Ethanol are added to 50 mL conical tubes in Section 5. Ensure resin in PhyTip columns are wetted with deionized water.

Protocols: Maxiprep


Gigaprep: Sample preparation All steps are carried out at room temperature. Please review the “Before Starting” section on page 8 prior to proceeding with Sample preparation.

Important reminder - Gigaprep samples are split in half to enable rapid processing. For example, if the total culture volume for a Gigaprep sample is 2 L, two 1 L cultures should be prepared and processed separately, using 1 buffer bottle per split sample.

1. Harvest Cells • Pellet a total of 1 – 3 L (0.5 – 1.5 L split) of an overnight bacterial culture by centrifugation at ≥

3500 x g for 30 minutes. Discard supernatant. • The MMG work deck can be set up while waiting for cells to pellet. See appropriate deck layout

per desired method for further instructions. 2. Resuspend Cells

• Uncap the centrifuge bottle and add the entire contents of BUFFER RB. To aid in pellet resuspension, agitation beads are included in BUFFER RB. Recap the centrifuge bottle and shake vigorously until bacterial pellet is completely resuspended.

Important reminders - Use 1 BUFFER RB bottles per split sample. Verify that RNase A is added to BUFFER RB.


• Uncap the centrifuge bottle and add the entire contents of BUFFER LB. Recap the centrifuge bottle and mix by inverting gently 20-30 times. To aid in lysis step, a blue indicator dye is present in BUFFER LB. Ensure the solution turns completely blue. If nonblue portions are observed after 30 inversions, continue mixing gently. Let the lysis step occur for at least 3 minutes. Do not let lysis step exceed 5 minutes. Important reminders - Use 1 BUFFER LB bottle per split sample. Verify that BUFFER LB has

not precipitated. Important notes


4. Precipitate • Uncap the centrifuge bottle and add the entire contents of BUFFER PB. Recap the centrifuge

bottle and mix by inverting 20-30 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 30 inversions, continue mixing gently. Important reminders - Use 1 BUFFER PB bottle per split sample. Important notes


5. Filter • Pour contents of centrifuge bottle into the middle of the SAMPLE FILTRATION PLATE.

Important reminders – Split samples are processed in SAMPLE FILTRATION PLATES 1A and 1B, or 1C and 1D.

Important notes - Care should be taken to avoid spilling and overflow. Do not add entire contents of centrifuge bottle into the SAMPLE FILTRATION PLATE if filtration occurs slowly. Wait until volume in SAMPLE FILTRATION PLATE has receded before adding the full contents.

Protocols: Maxiprep


Gigaprep kit sample preparation guide



Protocols: Gigaprep 1 Sample


Gigaprep: 1 sample method This protocol is designed for the preparation of up to 10 mg high-copy plasmid DNA using the AutoPlasmid MMG Gigaprep. Each Gigaprep sample is split in half when processed on the instrument. For example, if you had a culture volume of 3000 mL, 1500 mL would be used for each sample plate. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.

Culture volume range: 1 L— 3 L Culture volume recommended: 2 L


Deck Layouts: Gigaprep 1 Sample


Gigaprep: 1 sample method

Protocols: Gigaprep 2 Samples


Gigaprep: 2 samples method This protocol is designed for the preparation of up to 10 mg high-copy plasmid DNA using the AutoPlasmid MMG Gigaprep kit. Each Gigaprep sample is split in half when processed on the instrument. For example, if you had a culture volume of 3000 mL, 1500 mL would be used for each sample plate. While setting up the deck, place the appropriate consumables in the corresponding numbered section of the deck.

Culture volume range: 1 L— 3 L Culture volume recommended: 2 L


Deck Layouts: Gigaprep 2 Samples


Gigaprep: 2 samples method

Instrument Software Overview


Instrument Software Overview 1. Launch the PhyNexus MMG Software.

2. Select your desired method program for prep size and number of samples.

Prep & Sample Size Abbreviation Method time

Max

ipre

p

Maxi 1 Sample (1mL elution) Maxi 1 Sample (2mL elution) Maxi 1 Sample (3mL elution)

MX.1S.1E.V4 MX.1S.2E.V4 MX.1S.3E.V4

2 hours 25 minutes 2 hours 27 minutes 2 hours 29 minutes

Maxi 2 Samples (1mL elution) Maxi 2 Samples (2mL elution) Maxi 2 Samples (3mL elution)









Meg

apre

p

Mega 1 Sample (3mL elution) Mega 1 Sample (4mL elution) Mega 1 Sample (5mL elution)

MG.1S.3E.V4 MG.1S.4E.V4 MG.1S.5E.V4


Mega 2 Samples (3mL elution) Mega 2 Samples (4mL elution) Mega 2 Samples (5mL elution)









Gig

apre

p

Giga 1 Sample (6mL elution) Giga 1 Sample (8mL elution) Giga 1 Sample (10mL elution)

GG.1S.6E.V4 GG.1S.8E.V4

GG.1S.10E.V4


Giga 2 Samples (6mL elution) Giga 2 Samples (8mL elution) Giga 2 Samples (10mL elution)

GG.2S.6E.V4 GG.2S.8E.V4

GG.2S.10E.V4


3. Execute the method.

4. Once the method is completed, retrieve the plasmid DNA samples.



Navigating the software This section describes the details of the AutoPlasmid MMG software.

To open a Method, double-click on the PhyNexus MMG Software icon to launch it.

Figure 1: The AutoPlasmid MMG software as it appears when first opened. No method is currently loaded.

To select a specific method to run, click “file” —> “open” and select a “method”.

Figure 2: The AutoPlasmid MMG software as it appears when first opened. No method is currently loaded.



Navigate the subfolders to find the right method. Methods folder is located in: C:\PhyNexusHome\AutoController MMG SW\methods

Figure 3: Navigate the subfolders to find the right method.



Initializing the instrument

Before executing the opened method, it is necessary to initialize the instrument. Figure 3 displays the buttons associated with these steps. To properly initialize the instrument:

• Click the “Home” button. This will move the pipettes to the home position in the back right corner of the instrument.

• Click the “Reset” button. This will expel any excess volume in the pipette and bring the syringe to the home position.

Figure 4: The AutoPlasmid MMG software as it appears when a method is loaded. To initialize the

instrument, click the “Home” button (1), and click the “Reset” button (2).

Starting the method After the instrument is initialized, make sure that the deck is fully set up prior to starting the method. Once you are ready to begin: Click the “Method” tab —> click “Execute” and the instrument will begin running the selected method.



Once the method is completed, you will see the “Method execution complete” message in the lower left corner.

Once the method is completed, the plasmid DNA samples can be retrieved.

Troubleshooting


Troubleshooting This troubleshooting guide is designed to help solve most problems that may arise while using the AutoPlasmid MMG. The scientists at PhyNexus are always happy to answer any questions you may have about either the information or protocols in this manual. For questions not addressed here, please contact PhyNexus support to talk with a PhyNexus scientist. Problem & Cause Explanations and actions Problem Low DNA yield Cause Culture growth conditions

• Poor aeration of culture E. coli cultures need sufficient oxygen to grow for a prolonged time. Use a culture volume to flask size ratio of 1:5 or less. For best aeration, use a baffled culture flask with a vented or gas permeable seal.

• Antibiotic forgotten or insufficient Most antibiotics are light sensitive and degrade during long term storage at 2-8˚C or through multiple freeze-thaw cycles. For growth of overnight cultures use fresh antibiotic at correct concentrations.

• Plasmid did not propagate Bacteria culture may be too old. Check plasmid content in cleared lysate by running on a gel after performing an alcohol precipitation. Streak a fresh plate from a freezer stock. Inoculate antibiotic growth media with a freshly isolated bacterial colony from an overnight plate.

Problem Low DNA yield Cause Improper sample preparation

• Incomplete cell resuspension Pelleted cells should be completely resuspended before proceeding with lysis. After adding BUFFER RB to the pelleted cells, shake beads until the pellet is dislodged from the bottom of the vessel. Shake for an extra 10-20 seconds to ensure full suspension of the bacteria.

• Incomplete lysis Some bacteria cells are more resistant to lysis than others. Incubate suspended cells for 5 minutes after 30 slow inversions of the mixing bottle.

• Precipitation of BUFFER LB BUFFER LB stored below 20˚C will precipitate. Check BUFFER LB for SDS precipitation before use. If necessary, incubate the bottle for several minutes in a warm water bath. Mix well after incubation to ensure SDS is dissolved.

• Incomplete neutralization To completely precipitate SDS, chromosomal DNA, and other cellular debris after adding BUFFER PB, proper mixing is required. Incubating the lysate for 10-15 minutes after mixing BUFFER PB may help increase yield.

• Improper mixing vessel Use a large enough mixing bottle to ensure full mixing of buffers. A 500-mL bottle for Maxi, and a 1 L bottle for Mega/Giga are typically sufficient.

Troubleshooting


Troubleshooting

Problem Low DNA yield Cause Cell density of overnight culture

• The overnight culture density is too low Confirm that the cells were grown in optimal conditions. Increase the culture volume of the sample to be purified. Contact PhyNexus support for more information.

• The overnight culture density is too high Do not exceed the recommended pellet wet weights. Contact PhyNexus support for more information.

Problem Low DNA yield Cause Faulty wash buffer

• Ethanol not added to BUFFER WB or BUFFER WB(b). Plasmid DNA will elute from the PhyTip Column when BUFFER WB or WB(b) are used without adding the proper volume of ethanol.

• Ethanol evaporation. A lower ethanol percent in BUFFER WB or WB(b) will cause an abnormal amount of plasmid DNA to wash off the column. Ensure that the bottle cap is screwed on tightly after each use to prevent ethanol from evaporation from BUFFER WB and WB(b).

• Forgotten BUFFER WB in reservoir. Forgetting to empty the WB reservoir will cause the ethanol to evaporate from the forgotten volume. If new BUFFER WB is added to this volume, an improper ethanol percent could lead plasmid DNA to wash off the column. Always empty the WB reservoir after each run.

Problem Low DNA yield

• Elution volume too low. Use a higher elution volume for increased yield.

Troubleshooting


Troubleshooting

Problem & Cause Explanations and actions Problem Low DNA Quality Cause Improper sample preparation

• Incomplete neutralization. Incomplete neutralization generates poor quality supernatant. Ensure that neutralization is complete by inverting the sample 30 times. Do no vigorously mix, this may cause additional contaminant release from the flocculent.

• Ethanol contamination in eluate. If the PhyTip column is not fully dried prior to elution, ethanol may be present in the eluent. Ensure the vacuum trap is not full, and that the top is attached correctly.

• DNA is degraded. Make sure that equipment is kept as sterile as possible to limit nuclease contamination. Do not lyse for more than 5 minutes.

Problem Low DNA Quality Cause Improper sample preparation

• RNase A digestion was inefficient. Make sure to add RNase A to BUFFER RB. Store BUFFER RB at 2-8˚C if RNase A has been added and is not immediately used.

Problem Low DNA Quality Cause Improper sample preparation

• Overmixing of BUFFER LB or BUFFER PB. Shearing of genomic DNA can occur during overmixing. Do not vortex the mixing bottle during these steps.

Troubleshooting


Pipette Head Troubleshooting

Battery Check 1. Check if there is a battery installed inside each pipette by unplugging the USB cable and

sliding off the back cover. If the battery is present, replace the back cover. 2. If there is a battery inside, with the USB cable unplugged, check the battery level. There is a

battery icon at the top right corner of the screen. It should show as fully charged or close to fully charged.

3. Plug the USB cable back in. There will be a battery animation at the top right corner of the screen. It should be moving from right to left to indicate that it is charging. Once fully charged, battery animation will be full solid color without movement.

Computer Set Up 1. Go to Settings>System>Power & Sleep. Change Screen and Sleep, all to Never (for both

battery and plugged in). Close window. 2. Bottom left corner, there is a search box. Type “Close lid” and press enter. On the new

launched window, change settings to the following: When I press power button, “Shut down” for both on battery and plugged in. When I press sleep button, “Do nothing” for both battery and plugged in. When I close the lid, “Do nothing” for both battery and plugged in. Close window.

3. On the same search box, type “Power Option” and press enter. On the new launched window, mouse click “change plan settings”. Find and mouse click, “Change advanced power settings”. Another window will launch. On this window, scroll down to USB setting and change suspend setting to “Disabled” for both on battery and plugged in. Mouse click apply then OK.

4. On the same search box, type “Device manager” and press enter. On the new launched window, find and mouse click “Universal Serial Bus Controllers”. When expanded, double click on “USB Root Hub (xHCl)”. Another window will launch. On this window, select “Power Management” tab (top right). “Allow the computer to turn off this device to save power” setting, it should “not be checked”. Hit Ok.

Pipette QC Liquid Test 1. Hit the E-Stop. 2. Pull the pipette box forward. 3. Unplug the USB cable. 4. Put the pipette in Basic Mode by hitting the MAIN button > Select Basic by pushing in the

joystick/thumbstick.

Catalog Part Numbers


Catalog Part Numbers

Catalog # Description MMG-US AutoPlasmid MMG System - US voltage MMG-UK AutoPlasmid MMG System - UK voltage MMG-EU AutoPlasmid MMG System - EU voltage MMG-OT AutoPlasmid MMG System - OT voltage DPM 16-03-70 16 maxi-preps for AutoPlasmid MMG DPM 16-06-70 16 mega-preps for AutoPlasmid MMG DPM 08-06-70 8 giga-preps for the AutoPlasmid MMG

DPM 00-10-00 100 x 20 mL transfer tips for AutoPlasmid MMG

DPM 00-10-01 Sterile 100 x 20 mL transfer tips for AutoPlasmid MMG

DPM 00-20-00 50 conical tubes with caps for AutoPlasmid MMG, sterile, pyrogen free

DPM 00-20-01 50 conical tubes WITHOUT caps for AutoPlasmid MMG

DPM 00-30-00 AutoPlasmid MMG post-processing kit, Includes 16 each: 0.2 micron syringe filters, syringes and collection tubes

AutoPlasmid MMG User Manual v2 - PhyNexus · AutoPlasmid MMG Kits 3 AutoPlasmid MMG Instrument Deck...

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Transcript of AutoPlasmid MMG User Manual v2 - PhyNexus · AutoPlasmid MMG Kits 3 AutoPlasmid MMG Instrument Deck...