Page 1 of 11 Please refer to http://www.medicinalgenomics.com/product-literature/ for updated protocols and Material Safety Data Sheets (MSDS). Consult MSDS before using any new product. FEMINDICATOR is a registered trademark of Medicinal Genomics Corporation, and is for laboratory use only. Table of Contents Introduction... 1 Process Overview... 2 Kit Specifications... 3 Materials Supplied in the Kit... 3 Materials Supplied by the User... 3 Consumables & Hardware... 3 Reagents... 3 Decontamination Protocol... 4 Real- Time Quantitative PCR (qpcr) Protocol... 7 Glossary and Definitions... 11 LIMITED USE LABEL LICENSE... 11 Introduction FemINDICAtor utilizes a novel PCR based assay that is contamination free and provides an internal plant DNA control for every reaction. It is a simple two-step protocol, which is flexible and is automation compatible. FemINDICAtor plant gender detection kit uses a multiplexing strategy with an internal plant DNA reaction control to ensure accurate detection of plant gender for every reaction. Unlike other techniques, this multiplexing strategy verifies the performance of the assay when detecting gender, resulting in the minimization of false negatives due to reaction setup errors or failing experimental conditions.
Page 2 of 11 Process Overview 1. Use of restriction enzyme to digest the potential contaminant amplicon DNA. For more detail on this method see http://www.ncbi.nlm.nih.gov/pmc/articles/pmc4008621/ 2. Proceed directly to real-time quantitative PCR (qpcr) using a multiplex system of primers to detect the gender of the plant sample. Below is a simplified depiction of the qpcr assays. The forward and reverse primers have universal primer tails to enable potential Next Generation Sequencing of resulting products. Step%1:% Primers%and%probe%% bind%to%target%dna.%% Forward Primer Probe = Fluorophore = Quencher Reverse Primer Step%2:% PCR%occurs,%primers%are% extended%on%forward%and% reverse%dna%strands.%% Polymerization Probe Degradation Step%3:% Probe%is%degraded%as%a%result% of%polymerizaeon%and% fluorescent%signal%is% generated.%% Step%4:% Target%DNA%is%amplified%and% fluorescent%signal%can%be% measured%and%quanefied%.%% Result +" PCR Amplified DNA Fluorescent Signal
Page 3 of 11 Kit Specifications The qpcr Master Kit contains 100 reactions (Medicinal Genomics # 420002) or 500 reactions (Medicinal Genomics #420207) worth of the reagents. Each FemINDICAtor Plant Gender Detection Assay Kit contains 100 reactions worth of reagents. Each FemINDICAtor Positive Control contains 60 reactions worth of reagents. Materials Supplied in the Kit qpcr Master Kit V3, store at -15 to -20 o C upon arrival [Medicinal Genomics #420002]. Decontamination Enzyme (10 Units/µL) Decontamination Buffer (10x) qpcr Master Mix (5x) FemINDICAtor Plant Gender Detection Kit (Medicinal Genomics #420112) and Positive Control (Medicinal Genomics #420312), ordered separately, store at -15 o C to -20 o C upon arrival. Materials Supplied by the User Consumables & Hardware Bio-Rad CFX96 Touch TM Real-Time System (Medicinal Genomics #420203) Bio-Rad Personal PC (Medicinal Genomics #420204) 96 well optical qpcr plates (Bio-Rad Hard-Shell Low-Profile Thin-Wall 96-Well Skirted PCR Plates, Bio-Rad # HSP-9601 or Fisher Scientific 96-Well Armadillo PCR Plate, Fisher # AB2396) Adhesive optical seal for qpcr plates (Bio-Rad Microseal # MSB-1001 or USA Scientific TempPlate RT Optical Film # 2978-2100) Multi-channel pipette P50 or P20 (optional) Single channel pipette P10, P20 and P200 Filtered pipette tips for P10, P20, P50, and P200 Crushed ice or cold racks (96 well PCR Cryogenic Rack, VWR #89004-570 and 1.5ul Tube Bench-top Cryogenic Racks, VWR #89004-558 or similar) Freezer, -20 o C Table top mini plate centrifuge (Fisher Scientific #14-100-143 or similar) Table top mini tube centrifuge (VWR Mini Centrifuge #10067-588 or 6-place personal micro centrifuge for 1.5/2.0 ml tubes # 2631-0006, or similar) Table top Vortex Genie (Scientific Industries #SI-0236 or Similar) Reagents 10% bleach
Page 4 of 11 Decontamination Protocol 1. Begin with a 10% bleach wipe down of the workspace, including the bench top and all equipment being used (except the Bio-Rad CFX96 Touch TM Real-Time System and the Bio-Rad Personal PC). 2. Remove decontamination reagents and positive controls for the tested assays from the -20 o C freezer. 2.1. Decon Buffer and positive control Allow tubes to defrost at room temperature. Once defrosted, place tubes on ice. 2.2. Decon Enzyme Place directly on ice. 3. Before preparing the reaction, invert or vortex the tubes and spin-down the reagents. 3.1. Decon Buffer and Positive Control Vortex quickly followed by a pulse spin down in a micro centrifuge. 3.2. Decon Enzyme Invert the tube 5 times, followed by a pulse spin-down in a micro centrifuge. 3.3. Return all reagents to the ice. Do NOT vortex. 4. Begin with making 1Unit/µL stock of Decon Enzyme. 4.1. Label a 1.5ml tube 1:10 Decon Enzyme 4.2. Label a 1.5ml tube 1:10 Decon Buffer 4.3. Make a 1:10 dilution of Decon Buffer with H 2 O by adding 1ul of Decon Buffer into 9ul H 2 O. 4.4. Make a 1:10 dilution of 10 Unit/µL Decon Enzyme with previously diluted Decon Buffer. For every 1µL of 10 Unit/µL Decon Enzyme dilute with 9µL of diluted Decon Buffer. Note: It is best to add the largest volume reagent first, in this case Decon Buffer 4.5. Once combined, slowly tip mix or invert the tube 5 times. 4.6. Pulse spin-down in a micro centrifuge. 4.7. Return all reagents to the ice. 5. Make a master mix using the table below, labeling a 1.5ml tube as Decon MM Reagent 1 Reaction 24 Reactions (plus 2 48 Reactions (plus 4 excess rxn) excess rxn) Decon Buffer 0.75µL 19.5µL 39µL Diluted Decon Enzyme (1Unit/µL) 0.75µL 19.5µL 39µL H 2 O 1µL 26µL 52µL Total 2.5µL 65µL 130µL 5.1. The number of reactions must include a positive and negative control. For the positive control, make a 1:10 dilution. o 1ul of Positive Control (found in the kit) dilute with 9ul of H 2 O (found in the kit). For the negative control, use H 2 O (found in the kit). Note: It is best to add the largest volume reagent first, in this case the H 2 O. 5.2. Place the extraction plate on the magnet. 5.3. Use a new 96 well optical qpcr plate and label the plate Decontamination Plate_[date]. Add 2.5ul of Decon MM to each sample well, positive control well, and negative control well in the qpcr plate. 5.4. Remove the seal from the Extraction Plate and transfer 5uL of each sample into the corresponding well in the qpcr plate. Keep the extraction plate on the magnet when aspirating the 5uL. 5.5. Add 5uL of the diluted Positive Control to the corresponding well. Then add 5uL of water to the corresponding Negative Control well. Note: ALWAYS use a fresh tip for every liquid transfer into the qpcr plate. 5.6. Tip mix all wells by pipetting up and down 2 times. 6. Seal the plate with the adhesive seal, making sure to completely seal the plate wells using a pen or flat object to slide back and forth along the seal. 6.1. Spin down for at least 1 minute in the plate micro centrifuge. 6.2. Label the plate as Decontamination Plate [date]. 6.3. Place the sealed plate onto the Bio-Rad qpcr instrument, positioning the A1 well in the top left corner.
Page 5 of 11 7. Use the program labeled Decontamination on the Bio-Rad qpcr instrument. 7.1. Select User-Defined in the Startup Wizard under Run setup. 7.2. Use the Express Load dropdown menu to pick the Decontamination Program, and click Next. 1 hour at 25 o C, 20minutes at 65 o C followed by 8 o C forever.
Page 6 of 11 7.3. Close the lid and click Start Run. You will see the following warning message, Click OK. 7.4. Once the run has reached the 8 o C incubation you can stop the run at anytime by clicking the stop button. Then click OK on the subsequent confirmation.
Page 7 of 11 Real-Time Quantitative PCR (qpcr) Protocol 1. Remove qpcr Reagents and Assay Probe Mix tube from the -20 o C freezer. 1.1. qpcr Master Mix and Assay Probe Mix tube Allow tubes to defrost at room temperature. Once defrosted, immediately place tubes on ice. 2. Before preparing the reaction, invert or vortex and spin-down the reagents. 2.1. Assay Probe Mix tube Vortex quickly followed by a pulse spin-down in a micro centrifuge. 2.2. qpcr Master Mix Invert the tube 5 times, followed by a pulse spin-down in a micro centrifuge. 2.3. Return all reagents to the ice. Note: Do not vortex the qpcr Master Mix at any point during the protocol. 3. Make a master mix in a 1.5mL tube (the FemINDICAtor probe mix contains the internal plant control, SCCG probe mix). Note: It is best to add the largest volume reagent first, in this case H 2 O. Reagents 1 Reaction 24 reactions (plus 1 excess rxn) 48 reactions (plus 2 excess rxn) qpcr Master Mix 3.75µL 93.75µL 187.5µL Assay Probe Mix (Assay Specific) 1µL 25µL 50µL H2O 6.5µL 162.5µL 325µL Total 11.25µL 281.25µL 562.5µL 3.1. Once combined invert the tube 5 times to combine the Assay MM tubes 3.2. Pulse spin-down in micro centrifuge. 3.3. Place MM tube on ice until used in step 5. 4. Take the decontamination plate from the Bio-Rad qpcr instrument, and spin it down for 30 seconds in the plate micro centrifuge. 5. Remove the seal from the Decontamination Plate carefully (pay attention not to spill any liquid). Add 11.25µL of probe MM to each of the corresponding detection assay wells in the spun-down decontamination plate. 5.1. Tip mix all wells by pipetting up and down 2 times. 6. Seal the plate with the adhesive seal, making sure to completely seal the plate wells using a pen or flat object to slide back and forth along the seal. 6.1. Spin-down for at least 1 minute in the plate micro centrifuge. Note: Check for bubbles at the bottom of the wells (bubbles on the surface of the liquid is acceptable). If bubbles remain in the bottom of the wells, spin-down for another minute. 6.2. Re-label the plate as qpcr Plate_[date]. 6.3. Place the sealed plate onto the Bio-Rad qpcr instrument, positioning the A1 well in the top left corner.
Page 8 of 11 7. Use the program labeled qpcr Multiplex Detection on the Bio-Rad qpcr instrument. 7.1. Select User-Defined in the Startup Wizard under Run Setup. 7.2. Use the Express Load drop down menu to pick the qpcr Multiplex Detection Program and click Next. Hot start at 95 o C for 5minutes, followed by 40 cycles of 95 o C for 15 seconds and 65 o C for 90 seconds. 7.3. Design your plate under the plate tab in the Run Setup. Select the qpcr Multiplex Detection from the drop down menu. Click Edit Selected to move to the Plate Editor Screen.
Page 9 of 11 On the Plate Editor Screen. o Change the Sample Type to correlate with your specific plate setup. NOTE: To select the Sample Type, highlight the wells you would like to define, then choose from the drop down menu one of three types: Unknown Positive Control Negative Control o Double check that All Channels is selected from the drop down menu at the top. Note: This is the most important detail of the plate setup. The instrument needs to measure in all channels to complete the assay.
FemINDICAtor qpcr Plant Gender Detection Kit on the BIO-RAD CFX96 Touch Real Page 10 of 11 o Attach the flourophores to the wells being used.! Highlight all the wells being used! While the well locations are highlighted click to the on position both FAM and HEX When the plate is designed correctly click OK. o Click Yes to save your plate; if you do not save the plate, it will return to the default plate. o Save the plate. Note: Saving will override the template (that is fine). The template is used to make sure that All Channels is selected for Scan Mode. 7.4. Close the lid and click Start Run. 7.5. Save the experiment with the [User] and [date]
Page 11 of 11 Glossary and Definitions Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms. Polymerase Chain Reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. The Negative Controls are the samples where no Cq is expected. It helps to ensure that all Assay specific reactions are clean of contaminates. The Positive Control is the sample where a Cq is expected. It helps ensure that the FemINDICAtor Plant Gender Detection assay is working correctly. The gender specific Positive Control is targeting the male (Y) chromosome using the FAM flourophore. The Internal Control is added to every sample where a Cq is expected. It ensures the effectiveness and efficiency of each reaction. The internal control is targeting a Single Copy Control Gene or SCCG, using the HEX flourophore. LEGAL DISLCAIMER This test was developed as part of a research project conducted by Medicinal Genomics Corporation ( MGC ). Neither MGC nor any of their employees, contractors or other affiliates, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or any third party's use or the results of such use of any information LIMITED USE LABEL LICENSE This product is covered by at least one or more claims of US patents applications, which are exclusively licensed to Medicinal Genomics Corporation. This product is sold strictly for the use of the buyer, and the buyer is not authorized to transfer this product [or any materials made using this product] to any third party. 2015 Medicinal Genomics Corporation. All rights reserved. * All Trademarks are property of their respective owners.