GenPlate User Guide
Contents
1. Madisen L et al ME DNA banking the effects of storage of blood and isolated DNA on the integrity of DNA Am J Med Genet 1987 27 2 379 90 Schunemann HJ et al Short term storage of blood samples and DNA isolation in serum separator tubes for application in epidemiological studies and clinical research Ann Epidemiol 10 538 544 Ross KS et al Repeated freezing and thawing of peripheral blood and DNA in suspension effects on DNA yield and integrity J Med Genet 1990 27 9 569 70 Farkas DH et al Specimen stability for DNA based diagnostic testing Diagn Mol Pathol 1996 5 4 2 Adams M et al Rapid freezing of whole blood or buffy coat samples for polymerase chain reaction and cell culture analysis application to detection of human immunodeficiency virus in blood donor and recipient repositories The Transfusion Safety Study Group Transfusion 1993 33 6 504 8 GenTegra 16 GT20140402A w GenPlate GVN24P 4 shown Available GenPlate formats GVNP 20 Single region 384 element GenPlate maximum number of samples or maximum amount of sample GVN3P 20 3 region 96 element GenPlate GVN6P 20 6 region 40 element GenPlate well suited to DNA samples extracted from Buccal swabs GVN12P 20 12 region 16 element GenPlate well suited to DNA samples extracted from mouthwash collection GVN24P 20 24 region 4 element GenPlate most popular and commonly used format for human blood samples G enTeg ra 172. 0 GenPlate elements with buffy coat Note The pellet can be resuspended in as little as 200 ul to increase the guantity of DNA stored on a single element but will result in only 20 GenPlate elements spotted with buffy coat G enTegra 6 GT20140402A Blood Sample Handling Guidelines Before proceeding please read Crude Biosample Application Protocol page 4 Room Temperature Blood Storage Prior to Spotting 0 24 hours No change in DNA yield observed 24 72 hours Acceptable for use with GenPlate system but a slight decrease in yield may be observed 72 hours 8 days A steady decline in DNA yield is observed e 5 days 20 drop in yield e 8 days 33 drop in yield 2 8 weeks Not recommended for use with GenPlate system A considerable decline in the amount of recoverable DNA observed 4 C Blood Storage Prior to Spotting 0 24 hours No change in DNA yield observed 24 hours 8 days Acceptable for use with GenPlate system but a slight decrease in yield may be observed 2 8 weeks Not recommended for use with GenPlate system A considerable decline in the amount of recoverable DNA observed Frozen Blood Storage Prior to Spotting Low Temperature 20 C Results in an immediate drop in DNA yield compared to storage at 4 C This decline progresses steadily to a 30 40 drop in DNA recoverability after 1 year Ultra Low Temperature 80 C There is no influence on DNA yield or quality with long term storage years How
3. 1 Place 15ml of Scope mouthwash into your mouth 2 Swish mouthwash around your mouth for exactly 45 seconds During the swishing rub your cheeks gently against your teeth with your hands This will help increase the number of cells collected during the process 3 After 45 seconds of swishing and rubbing spit the content of your mouth into a 50ml conical tube 4 Proceed to Preparation of Cell Suspension from Mouthwash Samples Protocol page 13 Ensure that the mouthwash samples are processed and applied to GenPlates on the day of collection G enTeg ra 12 GT20140402A Preparation of Cell Suspension from Mouthwash Samples For best results process mouthwash samples and apply to GenPlates on the day of collection 1 Centrifuge the 50 ml conical vial containing the mouthwash sample for 5 minutes at 2000xg 2 Aspirate the supernatant off the cell pellet 3 Optional After removing most of the supernatant you may transfer the remaining liguid and pellet to a 1 7ml micro centrifuge tube centrifuge for an additional 5 minutes at 2000xg and remove the remaining supernatant using a pipette tip This helps to prevent dilution of the pellet in excess supernatant 4 Resuspend the cell pellet in 160 ul of 1X TE Buffer by vortexing at high speed and pipetting up and down It may be difficult to completely resuspend the cell pellet Pipetting up and down will help to break up viscous material If viscous material persists even afte
4. Application Protocol page 4 Additional Materials Reguired e Anticoagulant treated blood e 1 15 ml conical tube per sample e Ficoll Paque Plus e GE Healthcare Product codes 17 1440 02 amp 17 1440 03 e Salt Solution See Ficoll Paque Plus protocol for preparation instructions 1 1 Mix together 2 ml of anticoagulant treated whole blood and 2 ml of salt solution 2 Mix the Ficoll Paque Plus well and add 3 ml of it to a 15 ml conical tube 3 Carefully add the 4 ml of whole blood plus salt solution on top of Ficoll Paque Plus layer in the 15 ml tube Do not mix the two layers 4 Centrifuge sample at 400 x g for 30 40 minutes at 18 20 C Upon completion 4 layers should be visible From top to bottom plasma buffy coat Ficoll Paque Plus and granulocytes amp erythrocytes 5 Draw off the plasma layer using a clean Pasteur pipette and discard Be careful not to disturb the buffy coat layer 6 Using a clean pipette transfer the buffy coat layer into a clean 15 ml conical tube Be sure not to remove any Ficoll 7 Add 6 ml of salt solution to the conical tube containing the buffy coat and mix it up and down 8 Centrifuge the conical tube for 3 minutes at 2000xg A pellet should form 9 Aspirate the salt solution and discard GenTegra 5 GT20140402A 10 Re suspend the pellet in 400 ul 1x TE and proceed directly to the Crude Biosample Application Protocol on page 3 One 2 ml sample of whole blood will fill 4
5. GT20140402A Notes Page GenTegra s cra Notes Page GenTegra cra GenTegra
6. GenPlate User Guide Application of Crude Biosamples to GenPlates Version A April 2014 GenTegra SUPPORT GENTEGRA COM WWW GENTEGRA COM Application of Crude Biosamples to GenPlates for Whole Blood and Clones Blood Products Overnight Bacterial Cultures DMSO or Glycerol Bacterial Stocks Buccal Swabs Mouthwash Oragene DNA saliva G enTeg ra 2 GT20140402A Crude Biosample Application Protocol For preparation and application of the following types of crude biosamples to GenPlates for Whole Blood and Clones Blood products such as whole blood and buffy coat Overnight Bacterial Cultures DMSO Bacterial Stocks Glycerol Bacterial Stocks Buccal Swabs Mouthwash Oragene DNA saliva Before applying buffy coat please refer to Recommended Buffy coat Preparation een 5 For optimal recovery of DNA from whole blood please refer to the Blood Sample Handling Guidelines 7 Refer to the following supplemental protocols for preparation and application of the following types of crude biosamples Whole Blood a 4 DMSO or Glycerol Bacterial Stocks mnenus 8 Buccal Swab Samples ssssossosssosmassn ossia usa ve sven 9 Buccal Mouthwash Samples enerenasanenananes 10 Oragene DNA saliva sensed 15 Other Crude Biosamples eserenenunenananenanus 16 GenTegra 3 GT20140402A Crude Biosample Application Protocol Refer to this protocol for application of whol
7. GenPlates on the day of collection If swabs are not processed immediately storage at 4 C or drying overnight in a biosafety hood prior to room temperature storage may help to preserve DNA guality 3 Proceed to lysis protocol page 10 GenTegra 9 GT20140402A Lysing the Buccal Swab sample using GenSolve 1 Reconstitute Recovery Solution A with 16 ml of 1 0 LIDS solution provided Vortex to completely re suspend the lyophilized reagent 2 Pre heat incubator Shaker to 65 C Add 535 ul of Protease in the vial from step 1 and vortex briefly After addition of Protease Solution A should be used within 2 3 hours for maximum recovery Protease is typically yellow brown or orange in color 3 Cut the cotton swab from the stick With forceps place the buccal swab into a 2 0 ml micro centrifuge tube and add 300 yl reconstituted Solution A with Protease to the tube Make certain the swab is covered with liguid and at the bottom of the microtube Continue until each buccal swab sample has been processed 4 Incubate in the thermomixer set at 65 C and shaking at 1 400 rpm for 15 minutes 5 Add 10 ul Recovery Solution B to a new microtube and insert a spin basket Transfer the solution from Step 5 into the Spin Basket making sure to transfer along the swab by grasping with a forceps 6 Centrifuge at 16 300 x g for five minutes discard Spin Basket and swab Pulse vortex each microtube and centrifuge briefly 7 Proceed to Appli
8. cation Protocol Page 11 Refer to the GenSolve User Guide for details on the use of GenSolve for DNA extraction form biosamples GenTegra 10 GT20140402A Application of Lysed Samples to GenPlates For detailed application instructions refer to the Crude Biosample Application Protocol on page 4 1 Apply the entire volume of lysed buccal swab sample to elements on a Whole Blood GenPlate 10 ul per element The last element may have less than 10 ul A 6 region GenPlate may be practical for this sample type There is a maximum of 300ul of lysed sample which after digestion and centrifuging usually leaves 240 ul for spotting onto GenPlates A 6 region plate has 40 elements designated for each unigue sample The usual number of elements spotted per swab is 24 27 DNA Recovery 1 Follow the protocol in the GenSolve User Manual Whole Blood DNA Recovery from FTA paper Guthrie cards and GenPlate elements 2 Recover 6 elements in one tube at a time Six elements represent approximately 1 4 of the total buccal swab sample Typical DNA yields from human buccal swabs for 14 of the sample range from 0 1 1 5yg G enTeg ra 11 GT20140402A Buccal Mouthwash Samples Application Protocol Additional Materials Reguired e Scope Mouthwash e 50 ml conical tubes e TE Buffer e Centrifuge e Vortexer Collection of Mouthwash Samples This collection protocol is shown as an example your collection protocol may vary slightly
9. e Oragene DNA kit Application of Oragene amp DNA saliva Suspension to GenPlates For detailed application instructions refer to the Crude Biosample Application Protocol on page 4 1 Mix the Oragene DNA saliva sample in the Oragene DNA vial by inversion and gentle shaking for a few seconds 2 Incubate the sample at 50 C in a water incubator for a minimum of one hour or in an air incubator for a minimum of two hours 3 Apply the entire volume of sample to elements of a Whole Blood GenPlate 10 ul per element The heat treatment step is essential to ensure that DNA is adeguately released and that nucleases are permanently inactivated The incubation step may be performed at any time after saliva is collected and before it is purified Any volume of sample may be heat treated prior to GenPlate application Any remaining sample not applied to GenPlates can be stored at room temperature or frozen 15 C to 20 C The sample may be incubated at 50 C overnight if it is more convenient Refer to the instructions provided with the Oragene DNA kit for further information G enTeg ra 15 GT20140402A Application of other crude biosamples For additional guestions or information about applying biosamples to GenPlates which please contact GenTegra Technical Support at 1 925 461 3010 or support Agentegra com References Visvikis S et al DNA extraction and stability for epidemiological studies Clin Chem Lab Med 1998 36 8 551 5
10. e blood For preparation of other crude biosamples refer to the protocols on pages 4 16 prior to beginning this protocol 1 Remove the GenPlate from the foil pouch only when you are ready to use it The foil pouch is reusable for storing or shipping the plate 2 Remove and discard the silver adhesive seal on the top of the GenPlate before applying the sample to the storage element The GenPlate should be used within 60 minutes of removal from foil pouch or stored at ambient temperature and lt 40 relative humidity 3 Bring your sample to room temperature and rock for 10 15 minutes this applies to blood products only Spot 10 ul of the sample in the center of each well It is not necessary for the pipette tip to contact the storage matrix but the sample must be entirely dispensed onto the matrix to engage bactericidal and virucidal agents 4 After applying the sample or samples to a multi sample plate place the unsealed plate in a biosafety hood or an GenTegra Fast Dryer overnight 12 16 hours 5 After samples are dry apply the clear Adhesive Storage Plate Seal Ensure that the seal is firmly attached to the top of the plate avoid creating bubbles on the surface of the plate 6 Plate is now ready for storage To achieve maximum DNA yield allow the plate to cure for an additional 2 weeks before recovering DNA G enTeg ra 4 GT20140402A Recommended Buffy Coat Preparation Before proceeding please read Crude Biosample
11. ever short term storage 24 hours will alter DNA size G enTeg ra 7 GT20140402A DMSO or Glycerol Bacterial Stocks Application Protocol For detailed application instructions refer to the Crude Biosample Application Protocol on page 4 Typical Glycerol DMSO Stock Concentrations e DMSO Stocks are 10 DMSO e Glycerol stocks may range from 8 50 glycerol Preparation of the Glycerol DMSO Stock or bacterial culture 1 Depending on the downstream application for the DNA recovered from the glycerol stocks one ml of stock may be spun down to a cell pellet and resuspended in 250ul of LB TB SOC TE or TBS for a 4 fold concentration prior to application 2 Apply the entire volume of DMSO Glycerol Stock or bacterial culture to elements on a Whole Blood GenPlate 10 ul per element GenTegra 8 GT20140402A Buccal Swab Samples Application Protocol Additional Materials Reguired Incubator Shaker or comparable thermo mixer GVSPIN 250 Spin Basket Tube Assembly Micro centrifuge GVR 100 GenSolve for DNA recovery from whole blood spotted paper 100 recoveries e 2 0 ml micro centrifuge tubes Collection of Buccal Swab Samples This collection protocol is shown as an example your collection protocol may vary slightly 1 Place a cotton swab against either the right or left cheek and brush up and down from the rear of the cheek to the front 2 Place swab in a sealed collection tube For best results process swabs and apply to
12. r thorough vortexing and pipetting it may be discarded in a biohazard container prior to applying the remaining cell suspension to GenPlates Do not attempt to apply viscous filaments to GenPlates 5 Proceed to Application protocol page 14 GenTegra 13 GT20140402A Application of Mouthwash Cell Suspension to GenPlates For detailed application instructions refer to the Crude Biosample Application Protocol on page 4 1 Apply the entire volume of cell suspension to elements of a Whole Blood GenPlate 10 ul per element The last element may have less than 10 ul A 12 region GenPlate may be practical for this sample type There is a maximum of 160ul of lysed sample for spotting onto GenPlates A 12 region plate has 16 elements designated for each unigue sample The usual number of elements spotted per swab is 14 18 DNA Recovery 1 Follow the protocol in the GenSolve User Manual Whole Blood DNA Recovery from FTA paper Guthrie cards and GenPlate elements 2 Recover 1 6 elements in one tube at a time One element represents approximately 1 16 of the total mouthwash sample Typical DNA yields from human mouthwash for 1 16 of the sample range from 0 2 1 7 pg G enTeg ra 14 GT20140402A Oragene DNA saliva Application Protocol Collection of Saliva Samples Saliva collection is performed using the DNA Genotek Oragene amp DNA Self Collection Kit For proper collection of a saliva sample follow the instructions provided with th
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