Manual - Omega Bio-Tek
Contents
1. Samples are rich in protein Silica fine interference Make sure to completely vortex the sample with BL Buffer immediately Increase incubation time with BL Buffer and OB Protease Solution After applying to wells wash with 300 uL of a 1 1 mixture of BL Buffer and ethanol and then with DNA Wash Buffer Remove the silica fines by centrifugation and check the OD again 11 Troubleshooting Guide CEC Jo ETC Endonuclease Ba fe Ensure to wash the plate with HB Buffer Smeared DNA Contamination from gel p Remove the silica fines by centrifugation Silica fines interference and run the gel again CN fo Gowen Poor lysis for improper Mix thoroughly with BL Buffer prior to mixing with BL Buffer loading to the DNA plate No DNA eluted 100 ethanol not Before applying sample to column ethanol added to sample must be added as prescribed in protocol No ethanol added to Dilute Wash Buffer with the indicated DNA Wash Buffer volume of 100 ethanol before use pam foe Tem 3_ Incomplete Iysis due to Re Washing improper mixing with BL Buffer is viscous and the sample must be leaves colored B Buffer vortexed thoroughly residue in column Ethanol not added to Dilute Wash Buffer with the indicated DNA Wash Buffer volume of 100 ethanol before use 122. be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Store all other components at room temperature 22 25 C Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D1192 00 160 mL D1192 01 500 mL D1192 02 800 mL per bottle Before Beginning Yield and Quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold ofthe sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg ml A ratio greater than 1 8 indicates greater than 90 nucleic acid Alternatively quantity as well as quality can sometimes best be determined by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations Typically the majority of the DNA eluted is in monomeric supercoil form though concatamers may also be present Storage of Blood Samples Storage of blood samples without previous treatment leads to reduced yields of genomic DNA For the best result blood samples should be proceeded as following For short term storage up to a week collect blood in tubes containing EDTA as anticoagulant and store at 4 C For long term storage collect blood in tubes containing an anticoagulant and store at 70 C Thaw
3. uL Equilibration Buffer to each well Let sit at room temperature for 4 minutes Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Continue to Step 13 13 Transfer the samples from Step 11 to each well of the E Z 96 DNA Plate 14 Seal the E Z 96 DNA Plate with AeraSeal Film 15 16 17 18 19 20 21 22 23 24 25 26 27 E Z 96 Blood DNA Kit Protocol Centrifuge at maximum speed gt 3 000 x g for 5 10 minutes Make sure the samples have passed through the membrane Discard the filtrate and reuse the 96 well Square well Plate Remove and discard the AeraSeal Film Add 400 uL HB Buffer to each well Seal the plate with new AeraSeal Film Centrifuge at maximum speed for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Remove and discard the AeraSeal Film Add 700 uL DNA Wash Buffer to each well Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for instructions Seal the plate with new AeraSeal Film Centrifuge at maximum speed for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 22 26 for a second DNA Wash Buffer wash step 28 29 30 31 32 33 34 35 36 E Z 96 Blood DNA Kit Protocol Centrifuge the empty E Z 96 DNA Plate at maximum speed for 10 minutes to dry the plate Note This step is critical for rem
4. E Z 96 Blood DNA Kit D1192 00 1x 96 preps D1192 01 4x 96 preps D1192 02 20 x 96 preps September 2012 E Z 96 Blood DNA Kit Table of Contents Introduction and OvVervVie Wo coccion Kit Contents Storage and Stability Preparing Reagan Before Beginning ussesesensenssessenssessennssnnsenssenssnnssnnssnnsnene E Z 96 Blood DNA ProtoCO doi sconiastaarierns E Z 96 Vital DNA Pro Ol Troubleshooting Guides pis Manual Revision September 2012 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z 96 Blood DNA Kit allows rapid and reliable isolation of high quality genomic DNA or viral DNA from a wide variety of sample sources including fresh frozen or anticoagulated whole blood serum plasma bone marrow body fluids lymphocytes and cultured cells This kit incorporates the reversible nucleic acid binding properties of HiBind matrix in a high throughput 96 well format to eliminate proteins nucleases and other enzyme inhibitors or contaminators from blood or body fluids Up to 96 samples can be simultaneously processed in a single E Z 96 DNA plate The newly designed E Z 96 DNA plate has a binding capacity of 50 ug DNA per well Purified DNA is suitable for PCR restriction digestion and hybridization techniques There are no organic extractions thus reducing plastic waste and hands on time to allow up to 96 samples to be processed at one time If using the E Z 96 Blood DNA
5. Kit for the first time please read this manual in its entirety to become familiar with the procedures Blood or other body fluid samples are added to a specially formulated buffer containing detergent and mixed with proteinase Binding conditions are then adjusted and the sample is applied to the E Z 96 DNA plate Two rapid wash steps remove trace contaminants and enzyme inhibitors and pure DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition This manual has been edited for content and redesigned to enhance user readability OB Protease is now supplied in a liquid form eliminating the resuspension step to prior to use OB Protease Solution can also be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Kit Contents 96 well Square well Plate 2 2 mL a pf a f Note 96 well Square well Plates 2 2 mL can be used to collect filtrate from the E Z 96 DNA Plate They are designed for repeated use Wash the plates thoroughly in tap water after each use Let sit in 0 5M HCI at room temperature for 5 minutes Rinse with distilled water Used plates can also be autoclaved after washing Storage and Stability All components of the E Z 96 Blood DNA Kit are sguaranteed for at least 12 months from the date of purchase when stored as follows OB Protease Solution can
6. e the Before Beginning section on Page 4 for details Add 250 uL BL Buffer to each sample Take care not to touch the rims of the wells with the pipet tips which may lead to cross contamination Optional RNA will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 5 uL RNase A 20 mg mL 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 4 below 10 11 12 E Z 96 Blood DNA Kit Protocol Seal the 96 well Round well Plate with the Caps for Round well Plate Vortex or vigorously shake the plate side to side for 30 seconds to mix thoroughly Note Shake the rack side to side not up and down to prevent possible leakage around the caps Briefly centrifuge at 2 000 x g to collect any solution from the caps Incubate at 65 C for 10 minutes Rotate the plate gently once during incubation to mix Note Incubation at 65 C for more than 30 minutes can cause DNA degradation Briefly centrifuge at 2 000 x g to collect any solution from the caps Remove and discard the Caps for Round well Plate Add 250 uL 100 ethanol to each well Seal the 96 well Round well Plate with new Caps for Round well Plate Vortex or vigorously shake the plate side to side for 1 minute to mix thoroughly Briefly centrifuge at 2 000 x g to collect any solution from the caps Place the E Z 96 DNA plate on top of a 96 well Square well Plate 2 2 mL Optional Add 100
7. ed frozen blood sample at 37 C with gently agitation before use Sample Volumes Adjust the volume of each sample to 250 uL For samples smaller than 250 uL add the appropriate volume of PBS to bring the volume to 250 uL For samples larger than 250 uL split each sample into two 250 uL aliquots and use two wells of the 96 well Round well Plate 1 2 mL for Iysis The lysates can be recombined when transferring the samples to the E Z 96 DNA Plate E Z 96 Blood DNA Kit Protocol E Z 96 Blood DNA Kit Protocol Materials and Equipment to be Supplied by User Centrifuge capable of at least 3 000 x g equipped with swing bucket rotor Rotor adapter for 96 well plates Water bath heat block or incubator capable of 65 C 100 ethanol Multichannel pipet and nuclease free tips Optional RNase A stock solution 20 mg mL Before Starting Set water bath heat block or incubator to 65 C Prepare DNA Wash Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 65 C Add 25 uL OB Protease Solution into the bottom of each well of the 96 well Round well Plate 1 2 mL Add sample to each well of the 96 well Round well Plate Note Use 250 uL whole blood serum or body fluid for each well of the 96 well Round well Plate Up to 6 x 10 lymphocytes or cultured cells in PBS can be used in each well For sample volumes smaller or larger than 250 uL adjust the sample vol ume to 250 uL Se
8. oval of trace ethanol that may interfere with down stream applications Remove and discard the AeraSeal Film Incubate the E Z 96 DNA Plate at 65 C for 7 minutes to dry the plate Note These drying steps are critical for removal of trace amounts of ethanol that might otherwise interfere with downstream applications Place the E Z 96 DNA Plate on the 96 well Racked Microtubes Add 200 uL Elution Buffer heated at 65 C to each well of the E Z 96 DNA plate Let sit at room temperature for 2 4 minutes or in an incubator set to 65 C for 1 2 minutes Seal the E Z 96 DNA plate with new AeraSeal Film Centrifuge at maximum speed for 5 minutes Note First elution typically yields 60 70 of the DNA bound to the plate matrix A second elution with 200 uL Elution Buffer can increase the yield by 20 However increasing elution volume reduces the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 100 uL Elution Buffer Volumes lower than 50 uL greatly reduce yields Store DNA at 20 C E Z 96 Blood DNA Kit Protocol E Z 96 Blood DNA Kit Protocol Viral DNA 10 Integrated viral DNA or proviral DNA can be isolated by using the same standard protocol as for genomic DNA Viral DNA or RNA from extracellular viruses can be isolated with the E Z 96 Viral RNA Kit To avoid genomic DNA contamination acellular samples are recommended Use 10 12 ug carrier DNA such as poly dA o
9. r Poly dT for each 250 uL sample Adjust the binding conditions by adding 280 uL 100 ethanol instead of 250 uL at Step 9 of the standard protocol above Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Clogged well Low DNA yield Incomplete lysis Sample too large Add the correct volume of BL Buffer and incubate for specified time at 65 C It may be necessary to extend incubation time by 10 minutes If using more than 250 uL blood increase volumes of Protease BL Buffer and ethanol Pass lysate through one well successively lis sisas Divide sample into multiple wells adjust P volume to 250 uL with PBS Poor elution Improper washing Repeat elution or increase elution volume Incubation at 70 C for 5 minutes with Elution Buffer may increase yields Make sure the pH of the water is more than 7 5 DNA Wash Buffer must be diluted with 100 ethanol as specified on Page 4 Resin from the plate may be present in elu ate Avoid centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests Low A260 A280 ratio Extended centrifugation during elution step Poor lysis from improper mixing with BL Buffer Incomplete cell lysis n due to insufficient incubation
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