SPEEDTOOLS TISSUE DNA EXTRACTION KIT - Biotools

More documents

Recommendations

Info

F. Protocol for dried blood spots (e.g. Guthrie cards; NucleoCards; and FTA®cards)Before starting the preparation:• Check if Buffer BB3, Buffer BB5, and Proteinase K were prepared according to Section 4.• Set an incubator or water bath to 56ºC• Before elution, preheat Elution Buffer BBE to 70ºC.STEP DESCRIPTION1 PREPARE SAMPLECut one or two dried blood spots as accurately as possible. Cutspots into small pieces and place them in a 1.5 ml centrifuge tube.The area of the dried blood spots should be between 15 and 30mm 2 .2 PRE-LYSISAdd 180 µl Buffer BT1 and mix by vortexing. Spin the samplesbriefly and place them in a water bath or heating block and heat 10min at 94ºC. Let the sample cool down. Add 25 µl Proteinase K,vortex and incubate at 56ºC for 1h. Vortex occasionally duringincubation or use a shaking water bath.Be sure that the samples are completely covered with lysis buffer duringincubation.3 SAMPLE LYSISAdd 200 µl Buffer BB3 vortex vigorously and incubate at 56ºC for10 min.4 Proceed with Step 4 of the Standard Protocol.SAMPLE+180 µlBUFFER BT1Incubate 94ºC, 10 minCool+25 µlPROTEINASE KIncubate 56ºC, 1 hVortex200 µlBUFFER BB3VortexIncubate 56ºC, 10 minG. Protocol for hair rootsBefore starting the preparation:• Check if Buffer BB3, Buffer BB5, and Proteinase K were prepared according to Section 4.• Set an incubator or water bath to 56ºC• Before elution, preheat Elution Buffer BBE to 70ºCSTEP1 PREPARE SAMPLEDESCRIPTIONCut off the hair roots from the hair sample (up to 100) and collectthem in 1.5 ml collection tube.2 PRE-LYSISAdd 180 µl Buffer BT1 to the hair roots and freeze the samples inliquid nitrogen. Thaw samples in a 56ºC water bath. Repeat thisprocedure 4 times. Add 25 µl Proteinase K, mix by vortexing, andincubate 6-8 h or overnight at 56ºC. Use a shaking waterbath/incubator or vortex occasionally.HAIR ROOTS+180 µlBUFFER BT15 cycles of freeze/ thaw withliquid Nitrogen25 µlPROTEINASE KIncubate 56ºC,6-8 h /overnightVortex3 Proceed with Step 3 of the Standard Protocol.Ed 05 – March 12Page 9 of 19SPEEDTOOLS TISSUE DNA EXTRACTION KIT
H. Protocol from paraffin-embedded tissueBefore starting the preparation:• Check if Buffer BB3, Buffer BB5, and Proteinase K were prepared according to Section 4.• Set an incubator or water bath to 56ºC• Before elution, preheat Elution Buffer BBE to 70ºCSTEP DESCRIPTION1 PREPARE SAMPLEPrepare small sections (up to 25 mg) from blocks of fixed,embedded tissue. If possible, trim excess paraffin from the blockbefore slicing. Handle the sections with tweezers or toothpicks andplace the samples into microcentrifuge tubes.Add 1 ml n-octane or xylene to each tube. Vortex vigorously andincubate at room temperature for about 30 min. Vortexoccasionally. Centrifuge at 11,000 x g for 3 min. Pipette offsupernatant.Add 1 ml ethanol (96-100%) to each tube. Close and mix byinverting several times. Centrifuge at 11,000 x g for 3 min. Pipetteoff supernatant. Repeat the ethanol washing step. Pipette off asmuch of the ethanol as possible.Incubate the open tube at 37ºC until the ethanol has evaporated(~15 min).SMALL SECTIONS+1 ml n-OCTANEVortexIncubate RT, 30 min3 min, 11,000 × g+1 ml ETANOL3 min, 11,000 × g+1 ml ETANOL3 min, 11,000 × gIncubate open tubeat 37ºC2 Proceed with Step 2 of the Standard Protocol.I. Protocol for detection of Mycobacterium tuberculosis or Legionellapneumophila in sputum or bronchoalveolar lavageBefore starting the preparation:• Check if Buffer BB3, Buffer BB5, and Proteinase K were prepared according to Section 4.• Set an incubator or water bath to 56ºC• Before elution, preheat Elution Buffer BBE to 70ºC• Prepare N-acetyl cystein/NaOHFor this protocol depending on the number and the viscosity of the samples you may require and additionalbottle of Buffer BT1 (BIOTOOLS Ref. 21.161).STEP DESCRIPTION1 PREPARE SAMPLEAdd 200-500 µl sputum or bronchoalveolar lavage to an equalvolume N-acetyl cystein/NaOH 5 . Vortex gently to mix.Incubate the mixture for 25 min at room temperature with shaking.Adjust the volume to 25 ml with sterile water.Centrifuge for 30 min at 4,000 x g. Discard the supernatant.Resuspend the pellet in 0.5-1 ml Buffer BT1, depending onsample viscosity (Kit only includes 20 ml of Buffer BT1 6 ).Transfer 200 µl of the resuspended sample to a newmicrocentrifuge tube.ESPUTUM 200-500 µl+equal volN-acetyl cystein/NaOHVortexIncubate RT 25 minAdjust vol to 25 mlwith sterile H 2 O30 min, 4,000 × gResuspend pelletin 0.5-1 mlBUFFER BT1Transfer 200 µl2 Proceed with Step 2 of the Standard Protocol.5 N-acetil cystein/NaOH: 2 g NaOH; 1.45 g sodium citrate; 0.5 g N-acetyl cystein. Add water to 100 ml.6 Buffer BT1 Ref. 21.161Ed 05 – March 12Page 10 of 19SPEEDTOOLS TISSUE DNA EXTRACTION KIT
Page 1 and 2: Manufactured by:BIOTOOLS B&M Labs,
Page 3 and 4: 1. BASIC PRINCIPLESPEEDTOOLS TISSUE
Page 5 and 6: NOTEFor convenience, elution buffer
Page 7 and 8: 5 BIND DNAFor each sample, place on
Page 9: E. Protocol for yeastBefore startin
Page 13 and 14: 3-8 Proceed with Step 3 of the Stan
Page 15 and 16: N. Protocol for purification of gen
Page 17 and 18: 8. TROUBLESHOOTINGProblemPossible c
Page 19 and 20: 9. ORDERING INFORMATIONSPEEDTOOLS K

SPEEDTOOLS TISSUE DNA EXTRACTION KIT - Biotools

Create successful ePaper yourself

Delete template?

Save as template?