FavorPrep 96-well Genomic DNA Kit User Manual
Contents
1. FavorPrep 96 well Genomic DNA Kit User Manual Cat No FADWE 000 1 plate FADWE 001 4 plates FADWE 002 10 plates For Research Use Only v 1307 Introduction 96 Well Genomic DNA Extraction Kit is designed for high throughput purification of total DNA including genomic mitochondrial and viral DNA from whole blood and a variety of animal tissues or cells The method use proteinase K and a chaotropic salt guanidine hydrochloride to lyse cells and degrade protein then DNA in chaotropic salt is bonded to glass fiber matrix of plate After washing off the contaminanis the purified DNA is eluted by low salt elution buffer or water The entire procedure can be completed in one hour without phenol chloroform extraction and alcohol precipitation The kits can be used for manual filtration or with robotic handing systems and purified DNA with approximately 20 30 kb is suitable for PCR or other enzymatic reactions Quality Control The quality of 96 Well Genomic DNA Kit is tested on a lot to lot basis The purified DNA is checked byagarose gel analysis and quantified with spectrophotometer Kit Content FADWE000 FADWE001 FADWE002 1 plate 4 plates 10 plates FATG1 Buffer 23 ml 120 ml 250 ml FATG2 Buffer 23 ml 120 ml 250 ml W1 Buffer concentrated 33 ml 95 ml 95 ml x3 Wash Buffer concentrated 20 ml 50 ml 50 ml x3 Elution Buffer 23 ml 60 ml 240 ml Proteinase K 23 mg 90 mgt 225 mg tt 96 Well DNA binding plate 12. 5 minutes until wells have emptied Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Apply vacuum at 10 inches Hg for an additional 15 min or incubate at 65 C for 10 min to remove residual ethanol Add W1 Buffer to Add Wash Buffer each well of the to each well of the remove residual ethanol DNA binding Plate DNA binding Plate vacuum at 10 inches Hg vacuum at 10 inches Hg vacuum at 10 inches Hg 15 min Smin samin or incubate at 65 C 10 min STEP 4 NA Elution D Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And place the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH20 pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane completely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA j binding Plate 96 well PCR as pee E Plate A H 96 12 ml Add Elution Buffer Een art stand for 3 minutes 4 500 6 000 x g Plate 5 min 10 Centrifuge Protocol for Cultured Cells Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell Harves
3. 500 6 000 x g faa ped 5 mi DNA binding Plate DNA binding Plate in 21500 6 000 x g 15 min STEP 4 DNA Elution Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And place the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH2O pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane completely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA binding Plate 96 well PCR gt Plate 96 12 ml Add Elution Buffer MEREM stand for 3 minutes 4 500 6 000 x g Plate 5 min Vaccum Centrifuge Protocol for Tissue Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell lysis Add 200 ul FATG1 Buffer and 20 ul Proteinase K 10 mg ml to each well of a 96 Well 2 ml plate not provided Cut up to 25 mg of animal tissues or 0 5 cm of mouse tail and transfer into each well of 96 Well 2 ml plate Seal with Adhesive Film Incubate the plate with shaking at 60 C for 1 2 hours to lyse the sample If RNA free genomic DNA is required add 5 ul of RNase A 50 mg ml not provided to each well and incubate at room temperature for 4 minutes Add 200 ul FATG2 Buffer to
4. ml plate 2 0 ml 96 well deep collection plate 2 Centrifuge equiment with a swing bucket rotor capable of at least 5 000 X g 3 65 C and 70 C waterbaths or dry baths 4 Absolute 96 100 ethanol Centrifuge Protocol for Blood Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell lysis Add 200 ul FATG2 Buffer and 20 ul Proteinase K 10 mg ml to each well of a 96 Well 2 ml plate not provided Apply 200 ul of blood sample to each well and mix by pipetting Seal with Adhesive Film Incubate at 60 C for 20 minutes Preheat required Elution Buffer 50 100 ul per well at 60 C For Step 4 DNA elution Add FATG2 Buffer Add Blood sample Seal with adhesive film Incubate at 60 C and Proteinase K and mix by pipetting for 20 minutes STEP 2 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of another 96 Well 2 ml Plate not provided Transfer each well of the sample mixture to the 96 Well DNA binding Plate Place the assembly plates 96 Well DNA binding Plate 96 Well 2 ml plate in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate 96 well DNA binding Plate 96 well 2 ml Plate Add ethanol and Assemble plates Transf
5. pcs 4 pcs 10 pcs 96 Well PCR plate 1 pes 4 pcs 10 pcs Adhesive Film 2 pcs 8 pcs 20 pcs Add 100 200ml of ethanol 96 100 to each Wash Buffer when first use Add 12 35 ml of ethanol 96 100 to each W1 Buffer when first use Add 2 3 ml of ddH20 to the bottle and mix well store the prepared proteinase K at 4 C t Add 9 ml of ddH20O to the bottle and mix well store the prepared proteinase K at 4 C ttt Add 22 5 ml of ddH20 to the bottle and mix well store the prepared proteinase K at 4 C Specification Sample up to 200 ul of fresh frozen blood per well up to 25 mg_ of animal tissue up to 5X 10 animal cultured cells up to 10 bacterial cultured cells Binding Capacity up to 30 ug well Elution Volume 50 100 ul Operation centrifuge vacuum amp centrifuge Handling Time within 90 minutes Important Note 1 Buffers provided in this system contain irritants Wear gloves and lab coat when handling these buffers 2 The maxium sample size is described on Specification do not use the sample more than the limitation 3 Add required volume of ethanol 96 100 to Wash Buffer when first open 4 Add requred volume of ddH20 to proteinase K to prepare the 10 mg ml proteinase K solution and store the solution at 4 C 5 Prepare two dry baths or two water baths to 60 C and 70 C before the operation 6 Preheat the Elution Buffer to 65 C for DNA elution Additional Requirements 1 96 well 2 0
6. step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of another 96 Well 2 ml Plate not provided Transfer each well of the sample mixture to the 96 Well DNA binding Plate Place the assembly plates 96 Well DNA binding Plate 96 Well 2 ml plate in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate 96 well DNA binding Plate gt gt 96 well 2 ml Plate Add ethanol and Assemble plates Transfer the sample mixture mix by pipetting to the assemble plates iets 6 000 x g STEP 3 Warning Add 300 ul W1 Buffer to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for an additional 15 minutes to remove residual ethanol Add W1 Buffer to 4 500 6 000 x g Add Wash Buffer CN remove residual ethanol each well of the 5 min to each well of the 4
7. Centrifuge Protocol for Tissue Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell lysis Add 200 pl FATG1 Buffer and 20 ul Proteinase K 10 mg ml to each well of a 96 Well 2 ml plate not provided Cut up to 25 mg of animal tissues or 0 5 cm of mouse tail and transfer into each well of 96 Well 2 ml plate Seal with Adhesive Film Incubate the plate with shaking at 60 C for 1 2 hours to lyse the sample If RNA free genomic DNA is required add 5 ul of RNase A 50 mg ml not provided to each well and incubate at room temperature for 4 minutes Add 200 ul FATG2 Buffer to each well and mix by shaking Seal with Adhesive Film Incubate the plate with shaking at 70 C for 20 minutes until the sample lysate is clear Preheat required Elution Buffer 50 100ul per sample at 70 C For Step 4 DNA elution If there are insoluble material present following incubation centrifuge the plate for 5 minutes at full speed and transfer the supernatants to a new 96 Well 2 ml plate not provided Transfer tissue to each well of the Incubate the 2 ml plate 2 ml plate Seal with gt with shaking at 70 C adhesive film for 20 minutes Incubate the plate Add FATG1 Buffer nab anagat ee Add FATG2 Buffer f for 1 2 hrs and Proteinase K Seal with adhesive film mix by shaking STEP 2 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious
8. Plate Place the assembly plates 96 Well DNA binding Plate 96 Well 2 ml plate in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate 96 well DNA binding Plate gt gt 96 well 2 ml Plate Add ethanol and Assemble plates Transfer the sample mixture mix by pipetting to the assemble plates a 6 000 x g 11 STEP 4 Washing Add 300 ul W1 Buffer to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for an additional 15 minutes to remove residual ethanol Add W1 Buffer to 4 500 6 000 x g Add Wash Buffer QG remove residual ethanol each well of the 5 min to each well of the 4 500 6 000 x g DNA binding Plate DNA binding Plate 5 min 4 500 6 000 x g 15 min STEP 5 DNA Elution Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And pla
9. al ethanol Add W1 Buffer to Add Wash Buffer each well of the to each well of the remove residual ethanol DNA binding Plate DNA binding Plate vacuum at 10 inches Hg vacuum at 10 inches Hg vacuum at 10 inches Hg 15 min 5min zamin or incubate at 65 C 10 min STEP 5 DNA Elution Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And place the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH20 pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane completely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA binding Plate 96 wellPCR gt Plate 5 T qq H Add Elution Buffer 96 well 2 ml stand for 3 minutes 4 500 6 000 x g Plate 5 min 14
10. ate with shaking at 70 C for 20 minutes until the sample lysate is clear Preheat required Elution Buffer 50 100 ul per well at 70 C For Step 5 DNA elution Soa with i Incubate the 2 ml plate adhesive film a with shaking at 70 C Incubate the 2 ml plate for 20 minutes with shaking at 60 C for 20 min Add FATG1 Buffer and Proteinase K foe Sele ile fil Resuspend the pellet by mix by shaki aia pipetting sien i STEP 3 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of the vacuum manifold Transfer each well of the sample mixture to the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate Transfer the sample mixture to the assemble plates vacuum at 10 inches Hg Add ethanol and 5 min mix by pipetting 13 STEP 4 Washing Add 300 ul W1 Buffer to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Apply vacuum at 10 inches Hg for an additional 15 min or incubate at 65 C for 10 min to remove residu
11. ce the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH20 pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane completely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA binding Plate gt 96 wellPCR _ gt o Plate N 96 2ml Add Elution Buffer EET stand for 3 minutes 4 500 6 000 x g Plate 5 min 12 Vaccum Centrifuge Protocol for Cultured Cells Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell Harvesting Transfer the cultured cells to each well of a 96 Well 2 ml Plate not provided Cenirifuge at 1 000 x g for 10 minutes to pellet the cells discard the supernatant Step 2 Cell lysis Add 200 pl FATG1 Buffer and 20 ul Proteinase K 10 mg ml to each well of the 96 Well 2 ml plate and resuspend the pellet by pipetting Seal with adhesive film and incubate the plate with shaking at 60 C for 20 min to lyse the sample If RNA free genomic DNA is required add 5 ul of RNase A 50 mg ml not provided to each well and incubate at room temperature for 4 minutes Add 200 pl FATG2 Buffer to each well Seal with Adhesive Film and mix by shaking Incubate the pl
12. each well and mix by shaking Seal with Adhesive Film Incubate the plate with shaking at 70 C for 20 minutes until the sample lysate is clear Preheat required Elution Buffer 50 100ul per sample at 70 C For Step 4 DNA elution If there are insoluble material present following incubation centrifuge the plate for 5 minutes at full speed and transfer the supernatants to a new 96 Well 2 ml plate not provided Transfer tissue to each well of the 2 ml plate Seal with adhesive film Incubate the plate with shaking at 60 C Incubate the 2 ml plate with shaking at 70 C for 20 minutes Add FATG1 Buffer f for 1 2 hrs Add FATG2 Buffer ona Proteinase Seal with adhesive film mix by shaking STEP 2 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of the vacuum manifold Transfer each well of the sample mixture to the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate Transfer the sample mixture to the assemble plates vacuum at 10 inches Hg Add ethanol and 5 min mix by pipetting STEP 3 Washing Add 300 ul W1 Buffer to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for
13. ely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA binding Plate 96 well PCR Plate Add Elution Buffer 96 well 2 ml stand for 3 minutes 4 500 6 000 x g Plate 5 min Vaccum Centrifuge Protocol for Blood Sample Please Read Important Notes Before Starting The Following Steps Step 1 Cell lysis Add 200 pl FATG2 Buffer and 20 ul Proteinase K 10 mg ml to each well of a 96 Well 2 ml plate not provided Apply 200 ul of blood sample to each well and mix by pipetting Seal with Adhesive Film Incubate at 60 C for 20 minutes Preheat required Elution Buffer 50 100 ul per well at 60 C For Step 4 DNA elution Add FATG2 Buffer Add Blood sample Seal with adhesive film Incubate at 60 C and Proteinase K and mix by pipetting for 20 minutes STEP 2 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of the vacuum manifold Transfer each well of the sample mixture to the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Discard the flow through and return the 96 DNA binding Plate on top of the 96 Well 2 ml Plate Transfer the sample mixture to the assemble plates vacuum at 10 inches Hg Add ethanol and 5 min mix by p
14. er the sample mixture mix by pipetting to the assemble plates saa 6 000 x g STEP 3 Washing Add 300 pl W1 Buffer to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for 5 min Discard the flow through and return the 96 Well DNA binding Plate on top of the 96 Well 2 ml Plate Place the assembly plates in a rotor bucket and centrifuge at 4 500 6 000 x g for an additional 15 minutes to remove residual ethanol Add W1 Buffer to 4 500 6 000 x g Add Wash Buffer Cc remove residual ethanol each well of the 5 min to each well of the 4 500 6 000 x g DNA binding Plate DNA binding Plate 5 min 4 500 6 000 x g 15 min STEP 4 DNA Elution Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And place the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH20 pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane complet
15. ipetting ST EP 3 Washing Add 300 pl W1 Buffer to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Add 600 ul Wash Buffer ethanol added to each well of the 96 Well DNA binding Plate Apply vacuum at 10 inches Hg for 5 minutes until wells have emptied Apply vacuum at 10 inches Hg for an additional 15 min or incubate at 65 C for 10 min to remove residual ethanol Add W1 Buffer to Add Wash Buffer each well of the to each well of the remove residual ethanol DNA binding Plate DNA binding Plate vacuum at 10 inches Hg vacuum at 10 inches Hg vacuum at 10 inches Hg 15 min comin ES min or incubate at 65 C 10 min STEP 4 NA Elution D Place a clean 96 well PCR Plate provided on top of the 96 Well 2 ml Plate And place the 96 Well DNA binding Plate on the clean 96 Well PCR plate top 96 well DNA binding Plate middle 96 well PCR Plate bottom 96 Well 2 ml plate Add 50 100 ul Elution Buffer or ddH20 pH8 0 8 5 into the membrane center of the 96 Well DNA binding Plate Stand for 3 minutes until Elution Buffer or ddH20 has been absorbed by the membrane completely Place the assembly plates in a rotor bucket and centrifuge for 5 min at 4 500 6 000 x g to elute purified DNA 96 well DNA binding Plate 96 wellPCR _ en Plate au we Add Elution Buffer 96 well 2 ml stand for 3 minutes 4 500 6 000 x g Plate 5 min
16. ting Transfer the cultured cells to each well of a 96 Well 2 ml Plate not provided Centrifuge at 1 000 x g for 10 minutes to pellet the cells discard the supernatant Step 2 Cell lysis Add 200 ul FATG1 Buffer and 20 ul Proteinase K 10 mg ml to each well of the 96 Well 2 ml plate and resuspend the pellet by pipetting Seal with adhesive film and incubate the plate with shaking at 60 C for 20 min to lyse the sample If RNA free genomic DNA is required add 5 ul of RNase A 50 mg ml not provided to each well and incubate at room temperature for 4 minutes Add 200 ul FATG2 Buffer to each well Seal with Adhesive Film and mix by shaking Incubate the plate with shaking at 70 C for 20 minutes until the sample lysate is clear Preheat required Elution Buffer 50 100 ul per well at 70 C For Step 5 DNA elution Sea with r Incubate the 2 ml plate adhesive film with shaking at 70 C Incubate the 2 ml plate for 20 minutes with shaking at 60 C Add FATG1 Buffer for 20min Add FATG2 Buffer Seal with adhesive film mix by shaking and Proteinase K Resuspend the pellet by pipetting STEP 3 DNA Binding Add 200 ul ethanol 96 100 to each well of sample lysate in 96 well 2ml plate from precious step Mix immediately by pipetting 5 10 times Place a 96 Well DNA binding Plate on top of another 96 Well 2 ml Plate not provided Transfer each well of the sample mixture to the 96 Well DNA binding
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