Prep-Runner 96 Instructions

Instructions for Operation of the Prep-Runner 96 Items that you will need to start 1. Prep-Runner Instrument 2. Prep-Runner disposable rig trays 3. 12-well or 24-well cassette with Lysis Applicator Comb and Lysis Tablet Dispenser, or lysis buffers 4. 1 liter of 1X Running Buffer made in the plastic filling pail 5. 2% bleach solution ~500 ml Important: Follow the instructions in Appendix A for growth of bacteria in MR2001 broth. Figure 1. Loading Cassettes Step 1. Cassette Loading and Lysis: Wearing gloves, remove a 12 or 24-well cassette from its storage bag and place on its plastic bag. Remove the Applicator Comb from its bag and place in sample wells. Pipet 1.5 – 2.0 ml of bacterial culture for each growth tube into separate sample wells. Swish the Applicator Comb back and forth a few times. See figure 1. After approximately one minute, invert a Lysis Tablet Dispenser over the sample wells of cassette. Remove slider to one side allowing the tablets to fall into the individual wells. Swish the Applicator Comb back and forth a few times to stir the wells. Allow the culture to incubate with the Applicator Combs and dissolved Lysis Tablets while performing the steps below. Un-dissolved white powder binder from the lysis tablets will remain in the well bottoms even after the tablets have released their enzymes. Leave the applicator comb in place during the run. Make 1 liter of 1X Running Buffer from 100X stock using ultra-pure water (such as E-Pure, Milli-Q or double-distilled). 1X Running Buffer must be made fresh on the same day as the run. NOTE: The Prep Runner instrument gives optimal yield when its running buffer is between 22º and 30ºC. If the starting buffer temperature is below 22º C, the instrument will try to warm up the running buffer by applying higher voltage to its electrodes at the beginning of the run. This process will not add time to the run. Step 2. Set Up Instrument: Turn on instrument and select “Set-Up”. This menu allows you to set options for Cassettes, Machine, Lysis Time, and Program choice. Once these are set-up, select “Exit”, then select “Run”. Before starting program carry out the steps below.

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Step 3. Rig Tray Preparation: Rinse the tray briefly by filling with a ~2% solution of household bleach, then re-rinse tray with pure water and shake off water in sink. In addition, it is good technique to briefly dunk the electrophoresis wire cradle into the ~2% bleach solution in the rig tray and rinse this cradle prior to the run. Only one tray is required for 1-2 cassettes, a total of four cassettes can be processed with 2 trays. Place a disposable rig tray in one of the rig positions in the instrument. The symmetrical rig tray will come into register with the raised alignment bumps on the instrument’s internal floor. NOTE: A red temperature sensor button protrudes from the front right alignment bump. Use care when inserting the rig tray over this button. Step 4. Program Selection and Run: Place cassette(s) into rig trays with microchamber block facing the back wall of the instrument. Cassettes will fit between raised plastic ridges in rig tray. Replace electrophoresis wire cradle over cassette(s). The arrow on the wire cradle should point away from you as the operator. Notice the electrophoresis cradle will only fit over the cassettes in one orientation. Carefully pour 1X Running Buffer into the rig tray(s) using the one-liter plastic pail supplied with the instrument. Fill the rig tray up to the indent line on the side of the tray. The total volume is approximately 450-500 ml per tray when processing two cassettes per tray. Close the instrument’s lid. If the lysis time has not already been set, the display will prompt you to set the lysis time. 12 minutes is the default and a typical lysis time. The lysis time is the amount of time that the instrument waits before it begins the automatic run once the program is initiated in next step. The lysis time can be adjusted is the set-up mode using the right and left arrow keys. Some lysis time will have taken place during the previous steps. Select the run program from the instrument display. Program 1 is the standard plasmid purification run lasting 60 minutes, Program 2 is for plasmid purification (fast run) lasting 30 minutes, Program 3 is for plasmid > 7kb lasting 90 minutes, Program 4 is designed for a run in which microchamber blocks are manually pressed down by the operator (12-well cassette only), Program G1 is for genomic DNA, and program U1 allows the user to program the duration of voltage application steps. NOTE: If the ambient temperature of your lab is below 20ºC, it is advisable to run the plasmid purification over a 90-minute run. Allow the instrument to time its lysis step and automatically start, or press start to immediately activate run. The time remaining in the run is displayed. in well Step 4. Sample Removal: At the end of the run, remove cassette(s) from the instrument and place them on clean surface with the microchamber block facing toward you. Remove the 2030 µl of sample from each microchamber block well using a 200 µl disposable-tip pipettor, or use an 8-12 channel multi-tip pipettor to remove samples simultaneously. For optimal yields, you should remove the samples from block wells next to membrane the microchamber block within one minute of the end of the run. If final sample volumes are less than 20 µl or greater than 40 µl, refer to the trouble-shooting appendix. Remove rig trays and rinse with pure water. Place tray(s) inverted on a dry rack until further use. Rinse electrophoresis wire cradle with pure water, shake dry, and place back in instrument. Refer to Appendix A below for important information on culture growth, lysis time, and how the preparation works.

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Using the 24-Well Cassettes with the Prep-Runner 96 Instrument 24-well cassettes fit into the instrument and run on similar programs as the 12-well cassette. The microchamber block of the 24-well cassette is permanently fixed in the down position. 24–well cassettes hold only 1 ml of culture per lane and are supplied with liquid lysis buffers in place of applicator combs and lysis tablets. The liquid lysis buffers are added to the cassette wells using a 12-channel pipettor in every other lane per load. Load each lane of the 24-well cassette with culture using a single-channel or 12-channel pipettor. In order, add 100 µl of Lysis Buffer A, stir with the 12channell pipettor, wait 60 seconds, then add 20 µl of Lysis Buffer B to each lane from a separate clean reagent reservoir provided with 24-well cassettes. Stir the buffer into the culture using the multi-channel pipettor. Be certain that lysis occurs, as evidence by an increase in viscosity of the solution, before starting the run program. A numbered loading template is provided in each pack of 24-well cassettes that helps identify lanes during loading and un-loading. The yield of the 24 well cassette is typically 2/3 the yield of the 12-well version. NOTE: Programs 1, 2, or 3 of your Prep Runner instrument can be used for running the 24-well cassettes and will also work with 12-well cassettes. Failure to follow these instructions could result in blank lanes in the microchamber block. When using the 24 well cassette, it is important that the instrument is filled and drained only once. Removing Purified DNA Sample from a 24 Well Cassette

Reagent Reservoir

Samples can be removed from the 24-well cassette using either a single-channel pipettor or 12-channel pipettor. Since the 12-channel pipettor will remove every other lane, use the numbered loading template provided with your cassette pack to keep track of sample numbers and lanes.

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Appendix A. Guidelines for Plasmid Growth ESSENTIAL FOR GETTING GOOD YIELDS The Prep Runner-96 is designed to purify plasmid DNA directly from bacterial cultures of E. coli. The Prep Runner-96 yield is greatly affected by the amount of plasmid actually present in the E. coli culture. We have found that growth conditions of E. coli vary greatly depending on: 1) Proper aeration of the culture 2) Shake rate 3) Temperature of growth 4) Length of time of growth 5) The growth medium 6) Age of the streak/colony used to initiate a culture 7) Sterility of the culture 8) Use of fresh antibiotic in medium 8) Copy number of the plasmid The Prep Runner-96 separation process is sensitive to changes in growth conditions. Growth conditions that yield DNA with other purification kits and protocols may not give optimal results with this instrument. 1. Recommended Growth Conditions: a. Temperature/Time: 37˚ ± 1˚ C for 20 hours. Shorter growth time with MR-2001 will give lower yields. Note: MR-2001 does not contain anti-biotic when shipped. b. Shake Rate: 200-230 RPM in a gyratory environmental shaker c. Type of container and volume per flask: Always use one of the types of growth vessels with volumes as follows: 1. Round bottom disposable glass culture tubes (16 X 150 mm) with 2 ml or less of culture 2. Falcon 2059 or 2057 snap cap tubes (17 X 100 mm) with 2 ml or less of culture 3. 250 ml Erlenmeyer flask with 20 - 60 ml 4. Deep well microtiter plate growth blocks (with 2 ml square wells) containing only 1 ml of culture. Use the Mini-Grow 384 microtiter block shaker at a vigorous RPM to shake these deep well blocks. We recommend sealing the top of the blocks with lab tape, then puncturing holes in the tape to allow aeration during growth. Alternatively, microporous tape is available from MacConnell Research (Cat# MPT-100). THE VOLUME OF CULTURE PER GROWTH TUBE OR FLASK IS CRITICAL TO GETTING GOOD YIELDS WITH THE Prep Runner or Mini-Prep 96. In the data shown below, volumes ranging from 1 to 10 ml of culture were inoculated and grown identically in 15 ml (16 X 150 mm) glass disposable culture tubes for 20 hours at 37ºC. One ml of each culture was processed in a 2.0 ml cassette with the Prep Runner 96 instrument. The resulting purified plasmid DNA was run on a 1% agarose gel. The yield rapidly declines with an increase of culture volume per tube. 2 mls per tube gave optimal yields. Tube 1

2

3

4

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5

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Recommended Culture Growth Tubes: Best Good growth tube

Good

17 X 100 mm Falcon 2059/2057 Snap-Cap

Bad 15 ml vial

Grow plate in Mini-Grow 384 shaker

15 ml Falcon or similar tube The following strains of E. coli are recommended: Yield with 2.0 ml of Bluescript Plasmid in cell line E. coli cell line using C1202-SB cassettes 1. DH10B 3-6 µg 2. DH5aF’ 3-6 µg 3. DH5a 3-6 µg 4. DH5 3-6 µg 5. JM109 2-6 µg 6. HB101 3-5 µg 7. LE392 3-6 µg 8. MC1061 3-6 µg 9. INV1aF’ 3-6 µg 10. INV1a 3-6 µg 11. SURE 2-3 µg 12. DH11S 3-6 µg 13. TOP 10F’ 3-6 µg 14. TOP 10 3-6 µg 15. XL-1 Blue (1994) 3-6 µg 16. SOLR 0.1 µg 17. RR1 1 µg 18. BB4 2-6 µg * All DNA > 95% supercoiled Some bacterial strains contain nucleases that degrade plasmid DNA during the Prep Runner-96™ electrophoretic process. In general, these nucleases are excreted into the growth media and can be removed from the culture by concentrating the bacterial cells using centrifugation. Since the Prep Runner-96™ allows you to load bacterial culture directly, we recommend the use of the high yielding strains shown in the previous table. 2. Growth Medium We strongly recommend the use of Magnificent Broth (MR2001) with the Prep Runner-96 process. MR2001 has several features that result in maximal yields with the instrument process: (1) MR2001 is buffered so that even after growth of up to 24 hours, the culture is still at pH 7.8 to 8.0, which is compatible with the pH of the running buffer, process, and (2) MR2001 will allow the plasmid copy number to increase after the bacterial cells have reached a steady state in their growth cycle. This media produces higher DNA yields per volume than other media for this instrument. It will also produce excellent yields with other mini-prep protocols. Up to 5 ml of bacteria can be concentrated for loading into one cassette lane. Increasing the amount of bacterial cells will

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raise the yield up to three times that obtained from 1 ml. Growth media that give reasonable yields when cells are pelleted prior to addition of lysis buffers include: L-Broth, YT Broth, 2X YT Broth, and Super Broth. Terrific Broth does not provide good yields and will cause an overcurrent condition to occur with the instrument. 3. Age of the Bacterial Colony or Streak Begin culture inoculation with bacterial colonies or streaks that are less than two weeks old. The age of a bacterial colony or streak used to inoculate a liquid culture will affect the plasmid yield. Inoculations of a culture from a frozen glycerol stock will generally result in lower yields when compared to inoculation from fresh bacterial colonies. Never inoculate a liquid culture from another liquid culture. 4. Sterility of the Culture Inoculate cultures using sterile techniques. Antibiotics should be used at all stages of E. coli growth. Use antibiotic stock that is less then two months old. The Prep Runner-96™ purification procedure can be adversely affected by the sterility of the culture. A culture infected with foreign organisms will typically result in low yield and/or degraded plasmid DNA due to the presence of bacterial nucleases that arise from these foreign organisms. 5. Plasmid copy number We recommend the use of high copy number vectors as results obtained with low and medium copy number vectors can be low. The copy number of a plasmid in E. coli depends on: replication origin (ColE1, pMB1 or pSC101), insert size, and type of insert, among other factors. In general, plasmids can be grouped into three copy number categories: i) high copy number, such as pUC, pBluescript and their derivatives (300-650 copies per cell), ii) medium copy number, such as pBR322, pACYC, cosmids, etc. (2-20 copies per cell), and iii) low copy number, such as pSC101 and other single copy number vectors. In addition to the above, there are single copy large plasmids, such as PACs, YACs, and BACs that are used in molecular biology research. Refer to the subsequent procedures for a method to spin down and load 5-10 ml of cells for medium copy number vectors.

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Appendix B. DNA Sequencing with Prep Runner-96 Purified DNA Highly Active DNA from Starting Cultures Volumes as Low as 50 µ l a. Fluorescent Automated Sequencing DNA purified from the Prep Runner 96 is an excellent template for automated fluorescent sequencing using cycle sequencing protocols. Plasmid DNA taken directly from the instrument will give 900+ (base) sequencing reads on ABI 377, 3100, 3700, and 3730 instruments with amplified Big-Dye chemistries. The DNA taken directly from the PR96 will also sequence with Licor instruments. The following is a guide for the amount of PR-96 purified DNA to use in the automated fluorescent sequencing protocol. Automated Fluorescent Sequencing Signal Strength and Actual Read Length Examples Nanograms of PR-96/24 purified (4 kb) plasmid DNA Average Signal Strength Read Length* 1200 ng 2400 745 900 ng 2340 775 600 ng 230 828 300 ng 1890 828 240 ng 1420 872 160 ng 1300 821 80 ng 572 874 Nanograms of PR96 purified (18kb) plasmid DNA Average Signal Strength Read Length 50 ng 300-450 875 150 ng 350-500 855 * Read length is the base number of the sequence read before the first “N” appears.

Note: Extensive sequencing data shows that 1-2 microliters of the purified DNA, regardless of the yield, gives high quality sequence data. We do not recommend using more than 2 µl of the purified DNA in a sequencing reaction and, since the results show that less template gives better sequence reads, use of less than 2 µl of the purified DNA, (or ideally 1 µl) gives best results. This is due to the fact that less electrophoresis buffer salt is carried into the sequencing reactions with less volume of the purified DNA. DNA concentrations can be determined by fluorometry or by running a 2 µl aliquot of the purified DNA on a 1% TAE-agarose gel. The determination below illustrates that the highly active nature of the PR-96 DNA when used in fluorescent automated sequencing. In this trial, separate cassette lanes were loaded with: 2 ml, 1ml, 0.5 ml, 0.25 ml, 0.1 ml, and 0.05 ml of bacterial culture. Purification was carried out on program 1, then 2 µl of the resulting 20 µl of purified plasmid DNA was used for automated sequencing. The table below depicts the results. Starting Culture Volume Read Length Position of First “N” base read 2 ml (2 µl used for sequencing) 914 785 1 ml (2 µl used for sequencing) 915 786 0.5 ml (2 µl used for sequencing) 1050 766 0.25 ml (2 µl used for sequencing) 940 803 0.1 ml (2 µl used for sequencing) 980 732 0.05 ml (2 µl used for sequencing) 1060 765 CONCLUSION: Use 1-2 µ l of the instrument purified DNA for automated fluorescent sequencing, regardless of the yield. If automated fluorescent DNA sequencing does not work with DNA taken directly from the instrument, we recommend the following protocol. Precipitate 20-25 µl of the DNA with 2 volumes of ethanol and 0.1 volume of 2 M sodium acetate, then rinse this pellet with 70% ethanol prior to use in fluorescent sequencing protocols. b. Manual Sequencing Plasmid DNA prepared on the Prep Runner 96™ is highly active in manual DNA sequencing protocols and gives good quality sequence data. Note: You may find that plasmid DNA purified from bacteria grown in MR-2001 and processed on the Prep Runner 96 will have a slight yellow color. This yellow compound absorbs UV at 260 nm, and is visible as a light blue color on agarose or acrylamide gels stained with ethidium bromide and viewed on a UV transilluminator. This compound is a vitamin that co-purifies with the DNA and is not inhibitory to any enzymes, and is not toxic to mammalian cells. Yield measurements by OD260 will not be accurate.

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Appendix C. Genomic DNA Purification Genomic DNA User Protocol for the MP96 (Mouse Tail Snip, White Blood Cells, Tissue Culture Cells, and Bacteria) This protocol was used for the purification of mammalian genomic DNA from mouse tail snips. This same protocol can also be applied to genomic DNA isolation from tissue culture cells, dissected tissue, buffy coat and bacteria. Steps: 1. 0.25 – 0.75 mg of tissue is placed in a 1.5 mL snap-cap tube. 2. Add 100-500 ul of the digestion solution to the snap-cap tube and incubate at 55 C for 2 hours to overnight (until tissue is completely digested). Alternatively, the solution with tissue can be treated with a Bead Beater or Bead Bug device for 1 or less minutes, then loaded directly into the cassette’s cell. Digestion Solution: 0.1% SDS 50 mM Tris pH 7.5 10 mM EDTA 0.35 mg/mL Proteinase K 3. Centrifuge @ 13000 rpm for 15-20 min (this step may not be needed) 4. Remove supernatant and put in a clean 1.5 mL snap-cap tube. 5. 50 and 100 µl of the resulting supernatant was brought up to 500 µl and loaded into the sample wells of a 12 well cassette on the MP96 instrument. The microchamber block of the cassette was removed and discarded. Be sure the rig in the MP96 instrument is bleached and rinsed. 6. Prepare 2 Liters of 1X Running Buffer for this program. PROGRAM: Choose U1 for user input. The program is as follows: 30 min forward @ 100V 5 min reverse @ 100V 30 min forward @ 100V 5 min reverse @ 100V Buffer Change 30 minutes forward @ 100V 5 minutes reverse @ 100V End Program 7. After the program has ended and the rig has been drained, collect all of the samples directly from the starting sample loading wells. In some cases, you may discover that the final volume is slightly higher than your starting volume. 3-10 µl of the collected sample can be used directly in a 25 µl PCR reaction, or the entire sample can be ethanol precipitated to reduce the genomic DNA sample volume. Example of PCR reaction with Tail Snip genomic DNA purified by the above procedure. Figure 1. PCR of Tail Snip Genomic DNA HSP set 1 HSP set 2 HSP set 3 Lanes in the adjacent gel contain the PCR reaction from amplification of 7 µl (of 750 total recovered) of automatically purified mouse tail genomic DNA. Primer sets were designed to match mouse heat shock genes (HSP sets 1, 2, and 3). PCR reaction was performed on a MJ Research thermocycler for 30 cycles. Note: For other types of genomic DNA contact MacConnell Research Corp. 800-466-7949 [email protected]

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Mac Connell Research Price List Catalog Number Cassettes and Running Buffer C1202-SB C2402-SB G1202-120 C1500

Item Description

12-Well Sample Cassette, with applicator combs and Lysis Tablets (10 pack, 120 samples) 24-Well Sample Cassette, with lysis buffers (8 pack, 192 samples), Genomic DNA Cassette (10 pack - 120 samples) 100X Running Buffer Stock for MP-24 and 48, 250 ml

Enhance Growth Medium , Shaker, and Supplies MR2001-3 Enhanced Growth Media for Plasmids (300g) MR2001-10 Enhanced Growth Media for Plasmids (1 kg) MR2001-20 Enhanced Growth Media for Plasmids (2 kg) MG-384 Mini-Grow 384 Shaker MPT-100 Microporous tape, for sealing deep-well blocks during growth DWB-96 Deep Well Block (96 Well) for Bacterial growth (10 pack) DNA Size Markers EZM - 100 (high) Electrophoresis DNA Size markers - 200 bp to 10 kb (100 µg) EZM - 100 (low) Electrophoresis DNA Size markers - 100 bp to 1200 bp (100 µg) EZM - 250 (high) EZM - 250 (low)

Electrophoresis DNA Size markers - 200 bp to 10 kb (250 µg) Electrophoresis DNA Size markers - 100 bp to 1200 bp (250 µg)

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Price

$119

$189 $94 $20

$46 $149 $285 $3700 $20 $65 $58 $58 $245 $245