SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 1 of 8
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.
SENSATIVAX® is a registered trademark of Medicinal Genomics Corporation and is for laboratory use only.
Table of Contents
Introduction ..................................................................................................................................... 2 Process Overview ............................................................................................................................ 2 Kit Specifications ............................................................................................................................ 2 Materials Supplied in the Kit ......................................................................................................... 2 Materials Supplied by the User: .................................................................................................... 3 Extraction: ....................................................................................................................................... 4 Troubleshooting Guide: ................................................................................................................. 6 Glossary and Definitions ................................................................................................................ 7 LIMITED USE LABEL LICENSE................................................................................................. 7 Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 2 of 8
Introduction
SenSATIVAx® is a proprietary DNA isolation process that uses magnetic particles to isolate and purify both plant and
microbial DNA from a raw, homogenized plant sample. This approach is designed for ease of use and minimal
requirement of laboratory equipment. Large centrifuges have been replaced with lightweight mini-fuges, magnetic
particles, and magnets. The use of magnetic particles affords 8 tip or 96-tip automation, enabling both minimal entry costs
and high throughput applications. DNA can be isolated from a single sample or a large batch in under 1 hour. Hands-on
time is less than 45 minutes.
To enable minimal laboratory overhead, all organic solvents have been replaced with non-caustic reagents and 70%
EtOH. Magnet plates are available for purchase from Medicinal Genomics (part #420202).
Process Overview
= Plant and cellular materials
= Plant and Microbe DNA
= Magnetic particles
1. Add magnetic
particles to lysed
sample
2. Magnetic
particles bind
to DNA
3. Bound DNA is
separated from
sample by magnet
4. Wash with ethanol
to remove unbound
cellular components
5. Elute DNA in
aqueous buffer
6. Purified DNA is
transferred to new
container
Kit Specifications
The SenSATIVAx® Plant/Microbial DNA Purification Kit contains 200 reactions (Medicinal Genomics #420001) or 1000
reactions (Medicinal Genomics #420206) worth of reagents.
Materials Supplied in the Kit
•
•
•
o
o
MGC Lysis Buffer (Store at Room Temperature, 20 C to 28 C)
o
MGC Binding Buffer (Store at 2-8 C)
o
o
MGC Elution Buffer (Store at Room Temperature, 20 C to 28 C)
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 3 of 8
Materials Supplied by the User:
Consumables & Hardware:
• 96 well thermocycler or qPCR instrument
• Fisher Scientific 96-Well Armadillo PCR Plate, (Fisher # AB2396)
• 1X PBS diluted from 10X PBS pH 7.4 (American Bioanalytical #AB11072-0100 or similar)
• Integra 4.0mm Disposable Biopsy Punch with plunger (Integra # 33-34-p25 box of 25)
TM
• Kimwipes KIMTECH Delicate Task Wipers (#34133) or similar
• Premoistened Wipes 70% Isopropyl Alcohol, 30% Deionized Water (VWR Catalog # 21910-110)
®
®
• Adhesive optical seal for qPCR plates (Bio-Rad Microseal # MSB-1001 or USA Scientific TempPlate RT Optical
Film # 2978-2100)
• Multi channel pipettes P20 and P300, or P50 and P1000 (optional)
• Single channel pipettes P20, P200, & P1000
• Filtered pipette tips for P20, P50, P200, & P1000
• Table top mini plate centrifuge (Fisher Scientific #14-100-143 or similar)
Reagents:
• 10% Bleach
• Nuclease Free Water (American Bioanalytical # AB02124-00500)
• 70% Ethanol (EtOH) (American Bioanalytical product # AB00844-01000)
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 4 of 8
Extraction:
1. Begin with a 10% bleach wipe down of the workspace, including the bench top and all equipment being used.
o
2. Remove the MGC Binding Buffer from the 2-8 C refrigerator (allow it to come to room temperature before use).
3. On a clean surface lay down a Kimwipe
TM
or other disposable wipe.
4. Take the leaf to be tested and place it onto a clean wipe. Using a clean 4mm biopsy punch with plunger, firmly
press down on the leaf and twist the biopsy punch back and forth. Once the leaf has been punched transfer the
biopsied leaf to one well of a 96 well plate by pressing down on the biopsy plunger to dispense the leaf.
5. Clean the biopsy puncher before proceeding to the next leaf. Do this by wiping the metal biopsy part of the
puncher clean with a premoistened 70% Isopropyl alcohol wipe. Press the plunger into the wipe a few times to
ensure it is completely cleaned off. Be very careful not to cut yourself! Now you can proceed to the next leaf
sample. It is very important to clean the puncher in-between samples.
6. Once all of your leaf samples have been punched and transferred to a 96 well plate make the lysis master mix
using the table below.
Reagent
1 Reaction
1X PBS
MGC Lysis Buffer
Total
118.75µL
6.25µL
125µL
11 Reactions (plus 1
extra)
1.425mL
75µL
1.5mL
23 Reactions (plus 1
extra)
2.850mL
150µL
3mL
7. Vortex or tip mix the the lysis master mix until thoroughly mixed. Add 125µL of the lysis master mix to each well
of the 96 well plate containing the leaf samples.
8. 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.
o
9. Briefly spin the plate and then transfer to a thermocycler. Incubate the plate for 10 minutes at 95 C.
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 5 of 8
o
10. After 10 minutes at 95 C remove the plate, briefly spin plate and carefully remove the seal. If condensation has
formed on the top of the seal wait for the plate to fully cool and spin the plate before removing the seal.
11. Remove 100µL of the lysed sample and transfer to a fresh 96 well plate. If room in your plate permits, you can
transfer to a clean well on the same plate.
12. Vortex MGC Binding Buffer thoroughly. Check to make sure that the magnetic particles are completely
re-suspended in buffer before use.
13. Add 100µL of MGC Binding Buffer (this liquid is very viscous) to the 100µL sample, and pipette tip mix 15 times or
until homogenous.
a. Incubate the plate on the bench for at least 5 minutes.
Note: Be careful to avoid adding too many bubbles by pipetting gently when tip mixing. This is extremely
important as to not contaminate the wells in proximity.
14. Place the extraction plate onto the 96 well plate magnet plate for at least 5 minutes.
15. After 5 min incubation, remove as much of the 200µl of the supernatant as possible. Be careful not to disturb or
aspirate the beads.
a. Add 200µL of 70% ethanol (EtOH) with the extraction plate still on the magnet plate.
b. Wait at least 30 seconds, and remove all the EtOH.
Note: Take the pipet tip to the bottom center of the well to remove liquid.
16. Again, add 200µL of 70% EtOH with the extraction plate still on the magnet plate. Wait at least 30 seconds and
remove all the EtOH.
Note: If EtOH still remains in the wells, go back in with a smaller pipet tip to remove the excess.
17. After all the EtOH has been removed let the beads dry at room temperature on the magnet plate for at least 15
minutes. Be sure to remove all EtOH, as any leftover can inhibit qPCR results.
18. Remove the extraction plate from the magnet plate, and add 25µL of MGC Elution Buffer.
a. Tip mix approximately 15 times or until the beads are completely re-suspended.
Note: The re-suspensions may appear varied in their appearance, but the result will be the same.
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 6 of 8
b. Incubate the plate for at least 1 minute on the bench before returning the plate to the magnet plate.
c. Let the plate sit on the magnet for at least 1 minute before transferring the eluent to a new extraction
plate labeled with ”Final Extract [date]”.
19. Seal the plate with the adhesive seal, making sure to completely seal the plate wells using a pen or flat object to
o
slide back and forth along the seal. Store at -20 C until ready to perform qPCR protocol.
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 7 of 8
Troubleshooting Guide:
Symptom
Clumpy/Grainy Beads
Reason
Solution
Over-manipulation
of plant
Over manipulation of the plant can
cause the release of extra cellular
debris therefore clogging the beads with
extra material. To ensure this does not
occur, only use 1 finger of the leaf.
Insufficient time on
the magnet
Make sure the supernatant has fully
cleared before removing. Failure to do
so will result in bead loss, which will
result in DNA loss.
Bead Loss
Insufficient
pipetting
Extra elution volume
Insufficient removal
of Ethanol
Make sure no beads are aspirated
during supernatant removal; dispense
back supernatant, and attempt again
with a smaller volume after beads have
re-settled..
Make sure ALL ethanol is removed
before drying. This may require a
second or third aspiration. Carry-over
ethanol can cause inhibition in qPCR.
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com
SenSATIVAx® for FemINDICAtor™ Hole Punch Method
Plant Gender DNA Purification Kit
Page 8 of 8
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.
A supernatant is the liquid lying above the solid residue after centrifugation.
An eluent is a solution containing the DNA released from the MCG Binding Buffer.
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.
* All Trademarks are property of their respective owners.
Document EUD-00026 1.0
8 Cabot Road, Suite 2000 • Woburn, MA 01801 • 877.395.7608 • F 617.892.7191 • www.medicinalgenomics.com