Clean Plant DNA Kit User Manual
Contents
1. DNA lost during wash adding appropriate volume of ethanol prior to use Page 3 PGW3 Wash Buffer must be at Salt carryover Problems in room temperature downstream applications Dry the PRG Beads completely a ad before adding elution buffer Ordering Information Contact your local distributor to order Product Part Number Clean Plant DNA Kit 1 x 96 CP D0096 Clean Plant DNA Kit 4 x 96 CP D0384 CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com Notes CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com CLEAN NA CleanNA is a registered trade mark of GC biotech BV2. for instructions Place the plate on a magnetic separation device to magnetize the PRG Beads Incubate atroom temperature until the PRG Beads are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the PRG Beads Repeat Steps 20 23 for a second PGW3 Wash Buffer wash step Leave the plate on the magnetic separation device for 10 minutes to air dry the magnetic particles Remove any Residual liquid with a pipettor Remove the plate from the magnetic separation device Add 100 uL Elution Buffer heated to 65 C Vortex briefly or pipet up and down to resuspend the PRG Beads Incubate at 65 C for 10 minutes Place the plate on a magnetic separation device to magnetize the PRG Beads Incubate at room temperature until the PRG Beads are completely cleared from solution Transfer the supernatant containing the eluted DNA to a clean 96 well microplate not supplied Store DNA at 20C CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com CLEAN NA Please use this guide to troubleshoot any problems that may arise For further assistance please contact your local distributor Troubleshooting Guide Possible Problems and Suggestions For both fresh and frozen samples make sure to grind samples completely Incomplete disruption of starting material Decrease amount of starting Low DNA yield Poor lysis of tissue material Dilute PGW3 Wash Buffer by
3. species and tissues Up to ninety six 50 mg samples of wet tissue or 15 mg dry tissue can be processed in less than one hour The system combines CleanNA s buffer chemistry with the convenience of CleanNA s PRG Beaas to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates This kit is designed for manual or fully automated high throughput preparation of genomic chloroplast and mitochondrial DNA Purified DNA is suitable for PCR restriction digestion and hybridization applications There are no organic extractions thereby reducing plastic waste and decreasing hands on time to allow multiple samples to be processed in parallel Introduction and Principle The method will produce genomic DNA suitable for PCR and qPCR Because of the high throughput processing method high molecular weight genomic DNA may be sheared during lysis and may not be suitable for certain hybridization based assays or Southern blotting Plant samples are disrupted in a homogenizer bead based milling equipment PGL1 Buffer is added to lyse the sample Supernatant is then transferred to a new processing plate where PRG Beads are added to bind to the DNA Following a few wash steps DNA is eluted from the PRG Beads for downstream application Kit Contents and Materials Kit Contents Preparats me o romea Fones Materials and Reagents to be supplied by User Centrifuge capable of at least 3 000 5 000 x g Rotor ad
4. Catalog Nos CP D0096 CP D0384 Manual revision v1 01 Contents Introduction and Principle accurate 2 Kit Contents and Matelials une u 2 Preparation of Reagents sitiada oli leo 3 DNA Isolation from Fresh or Frozen Specimens coccccccccccccccccccnononanannncnnnnncnnnnnnnnnnnnnos 4 Troubleshooting Guide asada ctrasmcartresa detemcmaceng ten catnatonietpetinnd snot ccinatonecuatecageonias 6 Ordering Iniormallon soon e ee nO areca 6 A A A 7 For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice CLEANNA IS A REGISTERED TRADE MARK OF GC BIOTECH BV GC BIOTECH DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTEND ALLOWED BY LAW IN NO EVENT SHALL GC BIOTECH BE LIABLE WHETER CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTAL DAMAGES IN CONNECTION WITH OR ARRISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETER OR NOT FORSEEABLE AND WHETER OR NOT GC BIOTECH IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES TRADEMARKS The Trademarks mentioned herein are the property of GC biotech or their respective owners CLENA The Clean Plant DNA Kit allows rapid and reliable isolation of high quality genomic DNA from a wide variety of plant
5. apter for 96 well deep well plates Magnetic separation device for 96 well deep well plates 96 well deep well plates compatible with magnetic separation device Incubators capable of 56 C and 65 C Equipment for disrupting plant tissue Geno Grinder 2010 or MM300 Mixer Mill and tungsten carbide beads 8 or 12 channel pipette Reagent reservoir Sealing film Sealed deep well plate or capped microtube rack for sample disruption 100 ethanol Isopropanol CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com CLEAN NA Dilute PGW1 Buffer with 100 ethanol as follows and store at room temperature Preparation of Reagents PGW1 Buffer 100 Ethanol to be Added CP D0096 CP D0384 Dilute PGW2 Buffer with isopropanol as follows and store at room temperature PGW2 Buffer Isopropanol to be Added CP D0096 CP D0384 176 mL Dilute PGW3 Wash Buffer with 100 ethanol as follows and store at room temperature PGW3 Wash Buffer 100 Ethanol to be Added CP D0096 CP D0384 336 mL CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com CLEAN Clean Plant DNA Kit DNA Isolation from Fresh or Frozen Specimens The following method can be used for faster processing of samples For fresh samples this protocol may result in shearing of DNA For frozen samples this protocol may result in lower yields and sheared DNA Purified DNA is suitable for PCR and qPCR Before Star
6. card the cleared supernatant Do not disturb the PRG Beads Remove the plate from the magnetic separation device Add 500 uL PGW1 Buffer Vortex briefly or pipet up and down to resuspend the PRG Beads AN Note PGW1 Buffer must be diluted with 100 ethanol prior to use Please see Page 3 for instructions Place the plate on a magnetic separation device to magnetize the PRG Beads Incubate at room temperature until the PRG Beads are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the PRG Beads Remove the plate from the magnetic separation device CleanNA Phone 31 172 782 170 Web www cleanna com E mail info cleanna com CLEAN 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Add 500 uL PGW2 Buffer Vortex briefly or pipet up and down to resuspend the PRG Beads N Note PGW2 Buffer must be diluted with isopropanol prior to use Please see Page 3 for instructions Place the plate on a magnetic separation device to magnetize the PRG Beads Incubate atroom temperature until the PRG Beads are completely cleared from solution Aspirate and discard the cleared supernatant Do not disturb the PRG Beads Remove the plate from the magnetic separation device Add 500 uL PGW3 Wash Buffer Vortex briefly or pipet up and down to resuspend the PRG Beads AN Note PGW3 Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 3
7. ting Prepare PGW1 Buffer CSW2 Buffer and PGW3 Wash Buffer according to the instructions in the Preparing Reagents section on Page 3 Set an incubator to 56 C Heat Elution Buffer to 65 C Protocol 1 10 11 12 13 14 15 16 17 Grind 30 50 mg plant sample using a mechanical grinder such as Geno Grinder capped microtube rack in the presence of one or two grinding beads Process in the MM300 Mixture Mill or Geno Grinder Mixture Mill following the manufacturer s instructions A Note To prepare samples in 96 well plate format place samples in a sealed 96 well deep well plate or Add 500 uL PGL1 Buffer to each well Vortex to mix thoroughly Incubate at 56 C for 30 minutes Centrifuge at 4 000 x g for 10 minutes Carefully transfer 400 uL cleared lysate to a new 96 well deep well plate making sure not to disturb the pellet or transfer any debris k AN Note It is critical to leave the pellet undisturbed and avoid transferring debris as these can reduce yield Add 5 uL RNase A Vortex to mix thoroughly Incubate at room temperature for 10 minutes Thoroughly resuspend the PRG beads by pipetting or vortexing Add 400 uL isopropanol and 15 uL PRG Beads Vortex to mix thoroughly Incubate at room temperature for 5 minutes Place the plate on a magnetic separation device to magnetize the PRG Beads Incubate at room temperature until the PRG Beads are completely cleared from solution Aspirate and dis
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