Instruction Manual Whole Cell Yeast PCR Kit Rapid Isolation of DNA (Plasmid, YAC, and Genomic DNA) from Whole Yeast Cells (Colonies or Liquid Cultures) for PCR Analysis 200 Preps One Call One Source A World of Biotechnology Reagents Revision #: 2016-200-8G03P Catalog # 2016-200 Storage: ATTENTION! Refrigeration and Freezing is Required (-70 C RT) See pg. 4 Immediately Upon Delivery www.mpbio.com MP Biomedicals 29525 Fountain Parkway Solon, OH 44139 tel: 1.800.854.0530 fax: 1.800.334.6999
Whole Cell Yeast PCR Kit WHOLE CELL YEAST PCR KIT Specifications: 1-hour enzymatic reaction in PCR tubes or 96 well microplate followed by PCR. Release plasmid, YAC, and genomic DNA from yeast cells for PCR analysis. No phenol/chloroform extractions No alcohol precipitation No centrifugation Positive control (control yeast primers included to amplify genomic actin DNA) 200 Preps Revision #: 2016-200-8G03P Catalog # 2016-200 2 www.mpbio.com
TABLE OF CONTENTS 1. Introduction... 4 2. Kit Components... 5 3. Instructions Before Use... 5 4. Protocol... 7 5. Troubleshooting Guide... 10 6. Product Use Limitations & Warranty... 10 7. General Information... 12 www.mpbio.com 3
Whole Cell Yeast PCR Kit 1. Introduction The Whole Cell Yeast PCR Kit is designed to release DNA (plasmid, YAC, and genomic DNA) from whole cells (colonies or liquid cultures) for PCR analysis. Using a proprietary Enzyme Mixx, the tough yeast cell wall is disrupted in a single 1-hour incubation and the resulting cell suspension is used directly in the PCR reaction. Such a treatment is essential to give reproducible PCR results without purifying the DNA first. Optimal performance is obtained when Molecular Biology Certified yeast media from MP Biomedicals is used. This is particularly important if yeast liquid cultures are directly used without pelleting first. 4 www.mpbio.com
2. Kit Components Aurora Western Blotting kits are available in 2 sizes and sufficient for either 10 or 30 2016-201 Enzyme Mixx * 1 2016-202 Enzyme Buffer, Sterile 1.1ml 2016-205 Control Yeast BIO 96 Slant 2 2016-206 Control Actin PCR Primer Set (10uM) * * Store at -20 C. To reconstitute see instructions on Page 4. 1 Lyophilized Powder 2 Store at 4 C 3. Important Considerations Before Use 3.1 Shipping & Storage Instructions The kit is shipped at room temperature. However, upon receipt the Enzyme Mixx, Control Actin PCR Primer Set (both lyophilized powders) should be stored at -20 C. The slant containing the control yeast should be stored at 4 C. After the Enzyme Mixx is mixed with the Enzyme Buffer the resulting Whole Cell Lysis Solution should be stored at -70 C in single use aliquots. The reconstituted Control Actin PCR Primer is stored at -20 C. 3.2 Reagent Preparation Before first use, prepare the whole Cell Lysis Solution, Control Actin PCR Primer Set, and a frozen stock of Control Yeast. www.mpbio.com 5
Whole Cell Yeast PCR Kit I. Preparation of Whole Cell Lysis Solution a) Gently tap Enzyme Mixx vial on the bench top to collect the enzyme powder at the bottom of the vial. b) Resuspend Enzyme Mixx with the contents of the Enzyme Buffer tube (1.1 ml). Gently pipette up and down to mix. Place on a shaker or rocker and continue to dissolve at room temperature for ten minutes. Do not heat even though the solution may appear grainy. c) Aliquot in quantities that will be convenient for your testing needs. For example, if you expect to perform 10 PCR samples at once, freeze aliquots 50 ul (5 ul/ sample). d) Freeze aliquots at -70 C. To ensure maximum efficiency, thaw aliquots once and use entire contents. Do not refreeze. Enzyme is stable for 6-months after reconstitution. II. Preparation of Control Actin PCR Primer Set a) Spin tube containing primer set for 5 minutes at room temperature to collect the lyophilized oligos at the bottom of the tube. b) Add 140 ul of autoclaved H 2 O to get a 10 um stock solution. c) Spin for 10 minutes at room temperature. d) Store at -20 C. III. Preparation of a frozen stock of Control Yeast BIO 96 a) Inoculate a 5 ml YPD culture (MP Biomedicals: Catalog #: 4001-022) and grow overnight at 30 C with shaking. b) Transfer 800ml of culture to a sterile microfuge tube, add 200 l sterile 80% glycerol and invert several times to mix. c) Store at -70 C. d) To revive cells, scoop some frozen media from the frozen culture surface and streak onto a YPD agar plate (MP Biomedicals: Catalog #: 4001-222) and incubate at 30 C until growth is observed. Do not allow the frozen cells in storage vial to thaw. Call (440)-337-1200 or Toll Free: (800) 334-6999 for ordering information or visit 6 www.mpbio.com
www.mpbio.com 4. Safety Precautions Enzyme Buffer contains components that, when in contact with human tissue, may cause irritation. Wear personal protective equipment to prevent contact with the skin or mucus membranes (gloves, lab coat, and eye protection). Consult the enclosed Material Safety Data Sheet for additional details. 5. Protocol Note: To use the Control Yeast BIO 96 culture, streak a new YPD agar plate with control yeast each time. Optimal results are obtained with fresh colonies/cultures. If using liquid cultures, it is essential to use high-quality ingredients (Molecular Biology Certified yeast growth media is available from MP Biomedicals at www. mpbio.com) 1. For each sample to process, add 5 ul Whole Cell Lysis Solution to a microfuge tube, PCR tube, or a 96-well microplate well if processing multiple samples. 2. From prepared Yeast Colonies or Pelleted Cells: Lightly touch a fresh yeast colony or a cell pellet from a liquid culture with a sterile, disposable pipette tip. Transfer cell colony/pellet to the microfuge tube, PCR tube, or a 96-well microplate well containing the Whole Cell Lysis Solution. (Note: Transfer ~ ¼ of a 2 mm yeast colony). Pipette up and down gently or swirl with the pipette tip to resuspend. Avoid formation of bubbles. Cells should be a homogenous suspension for optimal results. www.mpbio.com 7
Whole Cell Yeast PCR Kit From prepared Yeast Liquid Cultures: Transfer 1 ul cells from an overnight culture to a microfuge tube, PCR tube, or a 96- well microplate well containing the Whole Cell Lysis Solution. Pipette up and down gently or swirl with the pipette tip to resuspend. Avoid formation of bubbles. Cells should be a homogenous suspension for optimal results. 3. Incubate at 37 for 1-hour and use the resulting cell suspension (NOT the clear supernatant) for a 25 or 50 ul PCR reaction. 4. Perform PCR assay (see guidelines below). To further lyse cells, include a 10-15 minute incubation step at 95 C as the first PCR step. 5. After running the PCR assay, spin each completed PCR sample for 2 minutes before loading an agarose gel in order to pellet cell debris at the bottom of the reaction tube. PCR cycling conditions to amplify genomic actin DNA from control yeast: 5 ul Control Yeast BIO 96 (processed above) 5 ul Control Actin PCR Primer Set 1 ul 200 mm dntps 5 ul 10X PCR PCR Reaction Buffer 2.5 ul 50 mm MgCl 2 0.25 ul Taq Polymerase (5U/ ul) Nuclease-Free H 2 O to 25 or 50 ul Note: Mix all reagents expect the Taq Polymerase, and incubate 10-15 minutes at 95 C. Next add 5 units of Taq Polymerase and begin 40 cycles of PCR. 8 www.mpbio.com
PCR Reaction Conditions: 95 C 10-15 minutes 95 C 45 seconds 50 C 30 seconds 72 C 1 minute 25 seconds Repeat for 40 Cycles 72 C 4 minutes 4 C Hold until use Expected PCR product size: ~700 bp Store at -20 C www.mpbio.com 9
Whole Cell Yeast PCR Kit 6. Troubleshooting Guide No PCR Signal? 1. Insufficient amount of yeast cells used. Upon resuspension of the transferred cells in whole cell lysis solution the resulting suspension should be somewhat cloudy. It should not be clear. 2. Cells with damaged cell walls may have settled to the bottom of the reaction tube; resuspend and use cell suspension in the PCR reaction (do not use the clear supernate which contains few cells). 3. Inefficient cell wall damage, thus no lysis occurs at 95 C. Increase the incubation time at 37 C in Step 3 and use fresh colony/culture. Call (440)-337-1200 or Toll Free: (800) 334-6999 for Technical Support or visit www.mpbio.com. 7. Product Use Limitation & Warranty Unless otherwise indicated, this product is for research use only. Purchase of MP Biomedicals products does not grant rights to reproduce, modify, or repackage the products or any derivative thereof to third parties. MP Biomedicals makes no warranty of any kind, expressed or implied, including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to, at our discretion, no replacement or compensation, product credits, refund of the purchase price of, or the replacement of materials that do not meet 10 www.mpbio.com
our specification. By acceptance of the product, Buyer indemnifies and holds MP Biomedicals harmless against, and assumes all liability for, the consequence of its use or misuse by the Buyer, its employees or others, including, but not limited to, the cost of handling. Said refund or replacement is conditioned on Buyer notifying MP Biomedicals, Inc. within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). www.mpbio.com 11
Whole Cell Yeast PCR Kit 8. General Information Ready-to-use Protocols For DNA, RNA and Protein Isolation From Any Sample Rapid and reproducible sample lysis and purification process No cross contamination with the closed lysing matrix tubes Increased yields of high quality DNA, RNA and proteins Integrity and size of DNA, RNA and proteins are retained Nucleic acids and proteins are ready-to-use in downstream application FastDNA Kit and FastDNA Spin Kit Cat N 6540-400 - Cat N 6540-600 respectively (100 preps) Lyse and isolate DNA in less than 30 minutes Plant, animal, yeast, fungal and microbial samples No hazardous organic reagents required SPIN filters streamline silica handling (FastDNA Spin Kit) FastDNA Spin Kit for Soil Cat N 6560-200 (100 preps) Lyse and isolate DNA in less than 30 minutes Variety of soil and environmental sample types No hazardous organic reagents required SPIN filters streamline silica handling FastRNA Pro Blue Kitt Cat N 6025-050 (50 preps) For use with gram positive and gram negative bacteria Lyse up to 1010 cells per 2ml tube Lysis and isolation with single-phase organic solution in less than 90 minutes FastRNA Pro Red Kit Cat N 6035-050 (50 preps) For use with yeast cells and fungal tissue Lyse up to 1010 cells per 2ml tube Lysis and isolation with single-phase organic solution in less than 90 minutes 12 www.mpbio.com
FastRNA Pro Green Kit Cat N 6045-050 (50 preps) For use with all plant and animal samples Lyse 50-100 mg tissue per 2ml tube Lysis and isolation with single-phase organic solution in less than 90 minutes FastRNA Pro Soil-Direct Kit and FastRNA Pro Soil-Indirect Kit Cat N 6070-050 - Cat N 6075-050 respectively (50 preps) Isolate RNA from soil samples (direct kit) and washed soil (indirect kit) in less than 2 hours Variety of soil and environmental sample types RNA protected during and after processing Humic acids reduced to allow uninhibited RT-PCR Includes additional reagents for even further purification if necessary SPIN filters streamline silica handling FastProtein Blue Matrix Cat N 6550-400 (50 preps) - Cat N 6550-500 (100 preps) Release of proteins from gram positive and gram negative bacteria in 40 seconds Protein extracts are ready for immediate electrophoresis or purification Ideal for optimizing induction conditions FastProtein Red Matrix Cat N 6550-600 (50 preps) - Cat N 6550-700 (100 preps) Release of proteins from yeast cells and fungi in 40 seconds Protein extracts are ready for immediate electrophoresis or purification Ideal for optimizing induction conditions www.mpbio.com 13
Whole Cell Yeast PCR Kit 14 www.mpbio.com
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