BiOstic Blood Total RNA Isolation Kit Sample (Catalog No S) (For isolation of total RNA from blood, buffy coat cells and bone marrow.

Transcription

1 BiOstic Blood Total RNA Isolation Sample (Catalog No S) (For isolation of total RNA from blood, buffy coat cells and bone marrow.) Information for Ordering Product Catalog No. Quantity Preps Instruction Manual Please recycle Version:

2 Table of Contents Introduction... 3 Protocol Overview... 3 Flow Chart... 4 Equipment Required... 5 Contents & Storage... 5 Precautions & Warnings... 5 Important Notes Before Starting... 6 Protocols: Experienced User Protocol... 7 Detailed Protocol (Describes what is happening at each step)... 9 Hints & Troubleshooting Guide Technical Guide Contact Information Other Quality Products Available

3 Introduction The BiOstic Blood Total RNA Isolation provides a way to purify RNA from up to 2 ml of fresh whole blood or 10 million cells. This method uses a simple hypotonic red blood cell (RBC) lysis to obtain a white blood cell (WBC) pellet for extraction of RNA without using phenol or ultra-centrifugation. RNase- Free DNase I is provided for on-column genomic DNA removal during the protocol, saving time and post-processing steps. Pure RNA is ready to use in downstream applications including RT-PCR, qrt- PCR, cdna synthesis, Poly A selection or RNA amplification. Protocol Overview BiOstic silica spin column products utilize the novel MO BIO Laboratories flat bottom spin column design, which provides improved sample processing and yields. The bucket configuration allows for enhanced sample flow and membrane drying after wash steps since the entire membrane is accessible to air flow. Silica technology provides a robust and fast way to purify nucleic acids without the use of organic solvents or cesium chloride gradients. The BiOstic Blood Total RNA Isolation begins with a single erythrocyte lysis using a hypotonic buffer that enables removal of the lysed RBCs after centrifugation. RNA is extracted from the remaining cell pellet using a highly denaturing chaotropic buffer that stabilizes and protects the RNA from degradation upon lysis. Vortexing is sufficient to homogenize and mechanically shear the genomic DNA to reduce viscosity and increase the affinity of RNA binding. To completely remove genomic DNA, RNase-Free DNase I is provided for on-column digest and one-step removal of both the enzyme and digested DNA with wash buffer. The purified RNA is ready to use in enzymatic reactions without additional treatments or handling steps. This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity UltraClean Lab Cleaner ml spray bottle 1 liter bottle RNase-Free Gloves 1556-XS 1556-S 1556-M 1556-L Bag of 150 Bag of 150 Bag of 150 Bag of 150 UltraClean Tissue RNA Isolation

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5 Equipment Required Microcentrifuge (10,000 x g) Pipettors Reagents Required but not Included β-mercaptoethanol Contents Component Amount Solution BR1 22 ml Solution BR2 1.6 ml Solution BR3 2 x 1 ml Solution BR4 1.8 ml Solution BR5 0.1 ml Solution BR6 2 x 1 ml Solution BR ml RTS DNase 15 µl Spin Filters 2 2 ml Collection Tubes 6 15 ml Collection Tubes 2 Storage Remove the tube of RTS DNase and store at 4 C. Store all other reagents and kit components at room temperature (15-30 C). The RTS DNase may be stored at 4 C for up to two years and at room temperature for up to 6 months without loss of activity. DNase is sensitive to physical denaturation. Do not vortex the resuspended DNase. Precautions Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact, wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact. All MSDS information is available upon request ( ) or at Reagents labeled flammable should be kept away from open flames and sparks. WARNING: Solutions BR3 and BR6 contain ethanol. They are flammable. 5

6 Important Notes Before Starting Not all blood samples are the same. Each person will differ in the number of white cells and this affects the amount of RNA you should expect from each sample. White cell count can vary based on the health of the person at the time of blood sampling. In general, RNA yields vary by species as well. An important consideration for extraction of RNA from blood is the handling and storage of samples. Blood should be collected in containing an anticoagulant such as EDTA, ACD, CPD-A or heparin. Extraction as soon as possible (within 2 hours) after blood collection will result in the highest quality and yields of RNA. Freezing of the samples should be avoided. Always use fresh blood for optimal results. To speed processing, prepare two 2 ml Collection Tubes per sample in advance and label the caps to make Spin Filter transfer more convenient. All steps in the protocol are carried out at room temperature. Mix Solution BR2 gently by shaking before use. Prepare Solution BR2 by adding β- mercaptoethanol (βme) Add 10 µl of β- mercaptoethanol (βme) for every 1 ml of the Solution BR2 for all samples to be processed. For each prep, 300 µl of BR2 will be needed. Note: Prepare Solution BR2 in smaller aliquots with fresh βme according to the number of samples you need to process that day instead of adding βme to the whole bottle. Use a fume hood when opening βme to avoid exposure to the chemical. Note: The lysis buffer containing βme will be stable at room temperature for one month. If you are starting with buffy coat, count the cells and begin at step 5 of the protocol. Use the amount of BR2 required for the number of cells in your sample according to the table on page 7. If starting with bone marrow, begin at step 1 of the protocol if the bone marrow appears bloody. If the bone marrow is clear of RBCs, begin at step 5. DNase I Solution Preparation and Storage The DNase provided in this kit is the RTS DNase, a room temperature stable DNase stored as a liquid. The concentration of the RTS DNase is 10 units/ µl. Before using, centrifuge the tube briefly to bring all of the DNase into the bottom of the tube. This enzyme should be stored at 4 o C for up to two years and can be kept at room temperature for up to 6 months without loss of activity. Additional RTS DNase may be purchased using catalog number To prepare the DNase I Solution, determine the volume of enzyme needed according to the number of samples. Per prep, combine 4 µl of RTS DNase I enzyme with 46 µl of Solution BR5. 6

7 Experienced User Protocol Please wear gloves at all times Use the following table as a guide for the correct volume of buffers to use for the lysis and binding steps to the RNA Spin Filter column. The protocol described below is written for starting with 500 µl of blood. Adjust your volumes according to your sample starting volume. Table 1. Volume of Blood Estimated number of WBCs* Volume of Solution BR1 Volume of Solution BR2 Volume Solution BR3 50 µl 2.5 x ml 300 µl 300 µl 100 µl 5.0 x ml 300 µl 300 µl 200 µl 1.0 x ml 300 µl 300 µl 500 µl 2.5 x ml 300 µl 300 µl 1000 µl 5.0 x ml 600 µl 600 µl 1500 µl 7.5 x ml 600 µl 600 µl 2000 µl 1.0 x ml 650 µl 650 µl * in healthy human blood Red Blood Cell Lysis: 1. Add 500 µl of blood to a 15 ml Collection Tube (provided) and add 2.5 ml of Solution BR1, cap the tube and invert to mix four times. Note: If you are processing less than 500 µl of blood, this first step may be carried out in a 2.0 ml Collection Tube (provided). 2. Incubate at room temperature for 10 minutes. Invert at least three times during this incubation. 3. Centrifuge at 2000 x g for 5 minutes to pellet the white blood cells. 4. Decant off the supernatant and invert the tube on a clean absorbent paper towel for at least a minute. Tap the tube a few times to absorb as much residual RBC lysate as possible. White Blood Cell Lysis: 5. Add 300 µl of Solution BR2 containing βme (see Important Notes Before Starting section) to the cell pellet and vortex on high speed for 1-2 minutes to completely homogenize the sample. If the sample is not completely resuspended, continue to vortex another minute. This step helps reduce genomic DNA contamination. Note: If you started with more than 500 µl of blood, increase the volume of Solution BR2 as directed in the Table 1 above. RNA Binding and Washing the Column: 6. Add 300 µl of Solution BR3 to the homogenized sample and mix thoroughly with pipetting. Note: The amount of Solution BR3 added should be equal to the volume of Solution BR2. 7. Transfer 650 µl of sample, including any precipitate, onto the Spin Filter column. Centrifuge at 10,000 x g for 30 seconds to bind. 7

8 8. Discard the flow through and put the Spin Filter back into the collection tube. A total of 2-3 loadings of sample may be required depending on your starting volume. Discard the flow-through and put the Spin Filter back into the 2 ml Collection Tube. 9. Add 400 µl of Solution BR4 to the Spin Filter column. Centrifuge at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter column into the same 2 ml Collection Tube and centrifuge at 10,000 x g for an additional 1 minute. This step will ensure the complete removal of Solution BR4 before the DNase I treatment. Note: Incomplete removal of Solution BR4 may inhibit the DNase activity. DNase I Treatment: 10. Transfer the Spin Filter to a new 2 ml Collection Tube (provided). To the center of the Spin Filter, add 50 µl of DNase I Solution (prepared by mixing 46 μl of Solution BR5 and 4 µl RTS DNase enzyme. See Important Notes before Starting section). Incubate at room temperature for 15 minutes. Note: Do not centrifuge the Spin Filter before the addition of Solution BR To the center of the Spin Filter column add 400 µl Solution BR4 and centrifuge the column at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter back into the 2 ml Collection Tube. Final Washes: 12. Add 500 µl of Solution BR6 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and place the Spin Filter back into the 2 ml Collection Tube. 13. Repeat the wash by adding another 500 µl of Solution BR6 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and place the Spin Filter back into the 2 ml Collection Tube. Spin the filter for 2 minutes at 10,000 x g to completely dry the membrane. RNA Elution: 14. Transfer the Spin Filter Column to a new 2 ml Collection Tube (provided). Add 50 µl Solution BR7 (Sterile Water) to the center of the Spin Filter. Incubate 1 minute then centrifuge the Spin Filter at 10,000 x g for 1 minute to elute. Note: A second elution with 50 µl may be performed to increase yields but will dilute the final concentration of RNA. Note: Placing Solution BR7 in the center of the small white membrane of the spin column will ensure that the entire membrane is wetted. This will result in a more efficient and complete release of the RNA from the silica membrane of the spin column. 15. Remove the Spin Filter and close the cap of the 2 ml Collection Tube. The RNA in the tube can be stored at -80 C until ready for use. Thank you for choosing the BiOstic Blood Total RNA Isolation. 8

9 Detailed Protocol (Describes what is happening at each step) Please wear gloves at all times Red Blood Cell Lysis: 1. Add 500 µl of blood to a 15 ml Collection Tube (provided) and add 2.5 ml of Solution BR1, cap the tube and invert to mix four times. Note: If you are processing less than 500 µl of blood, this first step may be carried out in a 2.0 ml Collection Tube (provided). What s happening: Solution BR1 is a hypotonic buffer that selectively lyses red blood cells (RBC s). 2. Incubate at room temperature for 10 minutes. Invert at least three times during this incubation. What s happening: Incubation and inversion facilitates complete RBC lysis and separation from white blood cells. 3. Centrifuge at 2000 x g for 5 minutes to pellet the white blood cells. What s happening: The white blood cells are separated after RBC lysis during centrifugation. 4. Decant off the supernatant and invert the tube on a clean absorbent paper towel for at least a minute. Tap the tube few times to absorb as much residual RBC lysate as possible. What s happening: RBC lysate is removed from the white blood cell pellet by decanting. White Blood Cell Lysis: 5. Add 300 µl of Solution BR2 containing βme (see Important Notes Before Starting section) to the cell pellet and vortex on high speed for 1-2 minutes to completely homogenize the sample. If the sample is not completely resuspended, continue to vortex another minute. This step helps reduce genomic DNA contamination. Note: If you started with more than 500 µl of blood, increase the volume of Solution BR2 as directed in the Table 1, page 7. What s happening: Solution BR2 is a highly chaotropic reagent required to completely lyse the white blood cells for RNA extraction. Solution BR2 also stabilizes and protects RNA from degradation. Vortexing mechanically shears genomic DNA and allows for better RNA binding efficiency. RNA Binding and Washing the Column: 6. Add 300 µl of Solution BR3 to the homogenized sample and mix thoroughly with pipetting. Note: The amount of Solution BR3 added should be equal to the volume of Solution BR2. What s happening: Solution BR3 is 70% ethanol and is used to create the conditions required for efficient binding of the RNA to the spin column while allowing proteins and cellular debris to pass through. 7. Transfer 650 µl of sample, including any precipitate, onto the Spin Filter column. Centrifuge at 10,000 x g for 30 seconds to bind. What s happening: RNA is bound to the spin column. 9

10 8. Discard the flow through. A total of 2-3 loadings may be required depending on your starting volume. Discard the flow-through and put the Spin Filter back into the 2 ml Collection Tube. What s happening: The flow through containing non RNA components is discarded. Multiple loads are required to process the entire sample volume through the filter. 9. Add 400 µl of Solution BR4 to the Spin Filter column. Centrifuge at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter column into the same collection tube and centrifuge at 10,000 x g for an additional 1 minute. This step will ensure the complete removal of Solution BR4 before the DNase I treatment. Note: Incomplete removal of Solution BR4 may inhibit the DNase I activity. What s happening: Solution BR4 is used to wash the spin filter column in preparation for the on column DNase I digestion. Complete removal of Solution BR4 is required for efficient and complete DNase I digestion. DNase I Treatment: 10. Transfer the Spin Filter to a new 2 ml Collection Tube (provided). To the center of the Spin Filter, add 50 µl of DNase I Solution (prepared by mixing 46 μl of Solution BR5 and 4 µl RTS DNase enzyme. See Important Notes Before Starting section). Incubate at room temperature for 15 minutes. Note: Do not centrifuge the Spin Filter before the addition of Solution BR4 What s happening: RTS DNase I is mixed with high activity digestion buffer and is used to completely remove genomic DNA from the column. 11. To the center of the Spin Filter column add 400 µl Solution BR4 and centrifuge the column at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter back into the collection tube. What s happening: Solution BR4 is a wash buffer used to inactivate DNase I and wash away residual enzyme and digested DNA while allowing RNA to remain tightly bound to the spin column. Final Washes: 12. Add 500 µl of Solution BR6 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and place the Spin Filter back into the 2 ml Collection Tube. 13. Repeat the wash by adding another 500 µl of Solution BR6 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow-through and place the Spin Filter back into the 2 ml Collection Tube. Spin the filter for 2 minutes at 10,000 x g to completely dry the membrane. What s happening: Solution BR6 is an ethanol based wash buffer used to remove residual salt and contaminants on the column in preparation for the release and elution of the bound RNA. Complete removal of all traces of the wash solution is critical. RNA Elution: 14. Transfer the Spin Filter to a new 2 ml Collection Tube (provided). Add 50 µl Solution BR7 (Sterile Water) to the center of the Spin Filter. Incubate 1 minute then centrifuge the Spin Filter at 10,000 x g for 1 minute to elute. 10

11 Note: A second elution with 50 µl may be performed to increase yields but will dilute the final concentration of RNA. Note: Placing Solution BR7 in the center of the small white membrane of the spin column will ensure that the entire membrane is wetted. This will result in a more efficient and complete release of the RNA from the silica membrane of the spin column. What s happening: Solution BR7 is highly pure water used to elute the RNA from the silica membrane of the spin column. 15. Remove the Spin Filter and close the cap of the 2 ml Collection Tube. The RNA in the tube can be stored at -80 C until ready for use. Thank you for choosing the BiOstic Blood Total RNA Isolation. 11

12 Hints and Troubleshooting Guide Spin Filter Clogging If excess of 10 million cells or 2 ml of blood is used, or if an inadequate amount of BR2 leukocyte lysis buffer was used for the lysis step, the spin filters have potential to clog. Count cells before starting if using buffy coat as a sample. If you are using non-human blood, use half the amount of blood recommended for the same volume of lysis buffers. If using whole blood from samples that may have elevated WBC count, scale back the volume of blood per column. To prevent clogging, increase the vortexing homogenization step another 2-3 minutes to shear genomic DNA and reduce viscosity. Centrifugation time for the sample loading step onto the spin filter can be increase from 30 seconds to 2 minutes. RNA Appears Degraded on Agarose Gels The BR2 buffer contains highly denaturing chaotropic salts to completely inactivate RNases. The use of Beta mercaptoethanol (βme) will destroy RNases and should be added fresh to Solution BR2. If RNA still appears degraded, the problem may be caused by the following: Make sure that blood samples are fresh and stored at 4 C if not processed immediately. Storage at either room temperature or -20 C will cause considerable RNA degradation and loss. Prepare Solution BR2 in smaller aliquots with fresh βme according to the number of samples you need to process that day instead of adding βme to the whole bottle. Homogenize thoroughly in BR2 making sure no cell clumps remain. The solution should be entirely homogenous. RNA will not always run correctly on non-denaturing gels and may appear smeared due to RNA secondary structure. Run RNA on a denaturing gel according to the Protocol for Formaldehyde Gel Electrophoresis. The 260/280 ratio is a good indicator of RNA quality as the absorbance at 260 will increase as RNA is digested into smaller fragments and single nucleotides. A ratio above 2.3 may indicate the RNA degradation. RNA Floats Out of Well When Loaded on a Gel Residual BR6 Wash Buffer may be in the final sample. To ensure complete drying of the membrane after BR6, centrifuge the spin filter in a clean 2 ml Collection Tube for an additional minute. Ethanol precipitation is the best way to remove residual Solution BR6. (See Concentrating the RNA below.) If you live in a humid climate, you may experience increased difficulty with drying of the membrane in the centrifuge. Increase the centrifugation times at step 15 by another minute. RNA has Low A 260/280 Ratio The ratio for pure RNA should be A 260/280 reading below 1.6 may have significant protein contamination. Make sure that the BR4 wash was performed before and after the DNase I treatment. A low ratio may also occur when the sample is measured by UV spectrophotometry in water. The low ph of water can influence the 280 reading and cause reduced sensitivity to protein contamination*. Re-measure the 260/280 diluting the RNA for measurement in 10 mm Tris ph 7.5. *Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of ph and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22,

13 Hints and Troubleshooting Guide cont. Genomic DNA Contamination in the RNA The BioOstic Blood Total RNA Isolation is provided with high quality RNase-Free DNase I for oncolumn digest. When used with the Solution BR5 included in the kit, activity of the DNase I will be optimal for on-column digestion. Use only the buffer provided with the DNase I for on-column digest. Make sure to perform the digest for the 15 minutes as recommended. Shortening the digest time may result in incomplete genomic DNA removal. RNA will not be degraded during this incubation. You may extend the DNase I digest up to 30 minutes. Homogenization of genomic DNA reduces the contamination on the column. Vortex vigorously in Solution BR2 to ensure thorough shearing of DNA. Concentrating the RNA Your final volume will be 50 μl μl. If this is too dilute for your purposes, add 5 μl of 3M Sodium Acetate and mix. Then add 2 volumes of 100% cold ethanol. Mix, and incubate at -70 C for 15 minutes or -20 o C for 2 hours to overnight. Centrifuge at 10,000 x g for minutes at 4 C. Decant all liquid. Briefly dry residual ethanol in a speed vac or ambient air. Avoid over-drying the pellet or resuspension may be difficult. Resuspend precipitated RNA in desired volume of RNase-Free water (Solution BR7). Storing RNA RNA is eluted in RNase-Free water (Solution BR7) and should be used immediately or stored at -20 C or -80 C to avoid degradation. RNA can be precipitated in ethanol and stored at -20 C to ensure minimal degradation during long term storage. 13

14 Technical Guide Protocol for Formaldehyde Agarose Gel Electrophoresis Solutions needed. 10x Formaldehyde agarose gel buffer 200 mm 3-[N-morpholino] propanesulfonic acid (MOPS) (free acid) 50 mm Sodium Acetate 10 mm EDTA ph to 7.0 with Sodium Hydroxide. 1x Formaldehyde agarose gel buffer (1L) 100 ml 10x Formaldehyde Agarose gel buffer 20 ml 37% (12.3M) Formaldehyde 880 ml DEPC treated water 5x RNA Loading Dye 16 μl Saturated aqueous Bromophenol blue solution 80 μl.5 M EDTA, ph μl 37% (12.3M) Formaldehyde 2 ml 100% Glycerol 3084 μl Formamide 4 ml 10x Formaldehyde agarose gel buffer Formaldehyde Agarose Gel preparation 1.2% in 100 ml Mix the following: 1.2 g Agarose 10 ml 10x Formaldehyde agarose gel buffer 90 ml DEPC treated water Heat the mixture in a microwave oven to melt the agarose. Cool to 65 C in a waterbath. Add 1.8 ml 37% (12.3M) Formaldehyde and 2 μl of 5 mg/ml Ethidium Bromide. Swirl to mix and pour into a gel box. The gel must be pre-ran for 30 minutes in 1x Formaldehyde Agarose gel buffer before loading the samples. RNA Sample Preparation The eluted RNA samples must be denatured before running on a formaldehyde agarose gel. To the sample, add 1 volume of 5x RNA loading dye for each 4 volumes of RNA sample i.e. 2 μl of 5x RNA loading dye for each 8 μl of RNA sample. Mix the samples and briefly centrifuge to collect the sample at the bottom of the tube. Incubate at 65 C for 3-5 minutes, then chill on ice and load in the Formaldehyde agarose gel. Run the gel at 5-7 V/cm in 1x Formaldehyde Agarose gel buffer. References 1. Beintema, J.J., Campagne, R.N., and Gruber, M. (1973). Biochim. Biophys. Acta 310: Kaplan, B.B., Bernstein, S.L., and Gioio, A.E. (1979). Biochem. J. 183:

15 Contact Information Technical Support: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA Ordering Information: Direct: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA For the distributor nearest you, visit our web site at 15

16 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. DNA Purification and Gel Extraction Catalog No. Quantity RNA Isolation Continued Catalog No. Quantity PowerClean DNA Clean-Up RNA PowerSoil DNA Elution preps Accessory UltraClean 15 DNA Purification preps RNA PowerSoil Total RNA Isolation preps UltraClean PCR Clean-Up UltraClean Microbial RNA Isolation UltraClean -htp 96 Well PCR Clean- Up x 96 preps 12 x 96 preps UltraClean Tissue & Cells RNA Isolation UltraClean GelSpin DNA Extraction UltraClean Plant RNA Isolation preps Plasmid DNA Isolation Catalog No. Quantity Genomic DNA Isolation Catalog No. Quantity UltraClean 6 Minute Mini Plasmid Prep PowerLyzer PowerSoil DNA Isolation UltraClean Standard Mini Plasmid Prep UltraClean -htp 96 Well Plasmid Prep UltraClean Midi Plasmid Prep x 96 preps 12 x 96 preps 20 preps PowerLyzer UltraClean Microbial DNA Isolation PowerBiofilm DNA Isolation PowerFood Microbial DNA Isolation UltraClean Maxi Plasmid Prep preps BiOstic Bacteremia DNA Isolation preps UltraClean Endotoxin-Free Mini BiOstic FFPE Tissue DNA Isolation Plasmid Prep UltraClean Endotoxin-Free Midi preps BiOstic Paraffin Removal Reagent x 25 ml Plasmid Prep UltraClean Endotoxin-Free Maxi preps PowerMax Soil DNA Isolation preps Plasmid Prep UltraClean Endotoxin Removal kit PowerSoil DNA Isolation UltraClean Endotoxin-Free Ethanol Precipitation kit PowerSoil -htp 96 Well Soil DNA Isolation x 96 preps 12 x 96 preps UltraClean Endotoxin Removal Reagent ml UltraClean Soil DNA Isolation Endotoxin-Free Sodium Chloride ml UltraClean -htp 96 Well Soil DNA Isolation x 96 preps 12 x 96 preps Endotoxin-Free Centrifuge Tubes each/2 ml UltraClean Mega Soil DNA Isolation preps each/15 ml 25 each/50 ml RNA Isolation Catalog No. Quantity PowerClean DNA Clean-Up PowerWater RNA Isolation (No Filters) NF UltraClean Fecal DNA Isolation PowerLyzer UltraClean Tissue & Cells RNA Isolation PowerLyzer UltraClean Plant RNA PowerMicrobial Midi DNA Isolation preps Isolation PowerBiofilm RNA Isolation PowerMicrobial Maxi DNA Isolation preps LifeGuard Soil Stabilization Solution ml 1 L UltraClean Microbial DNA Isolation On-Spin Column DNase I (RNase- Free) BiOstic Stabilized Blood RNA Isolation BiOstic Blood Total RNA Isolation UltraClean -htp 96 Well Microbial DNA Isolation PowerPlant DNA Isolation UltraClean Plant DNA Isolation x 96 preps 12 x 96 preps 2 16

17 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Genomic DNA Isolation Continued Catalog No. Quantity UltraClean -htp 96 Well Plant DNA x 96 preps Isolation x 96 preps UltraClean Tissue & Cells DNA Isolation UltraClean -htp 96 Well Tissue DNA x 96 preps Isolation x 96 preps UltraClean Blood DNA Isolation (Non-Spin) UltraClean Blood DNA Isolation (Processes 1,000 ml of Blood) UltraClean Blood DNA Isolation Plus RNase (Processes 1,000 ml of Blood) UltraClean BloodSpin DNA Isolation UltraClean -htp 96 Well BloodSpin DNA Isolation UltraClean Forensic DNA Isolation PowerWater Sterivex DNA Isolation PowerWater DNA Isolation (No Filters) RapidWater DNA Isolation (No Filters) UltraClean Water DNA Isolation (No filters) Other Reagents and Lab Accessories Catalog No. Quantity 20 bp DNA Ladder µg 100 bp DNA Ladder µg UltraClean Agarose, Molecular Biology Grade 1 kb DNA Ladder µg kit UltraClean MS-8 Agarose kit UltraClean Forensic Agarose x 96 preps 12 x 96 preps UltraClean Low Melt Agarose UltraClean Low Melt Sieve Agarose Ethidium Bromide Solution isolations 20 isolations NF Ethidium Bromide Destaining Tea Bags NF Dye Dots Dry Gel Loading Dye NF with Bromophenol Blue NF Bromophenol Blue Gel Loading NF Buffer NF 10 preps Bromophenol Blue/Xylene Cyanol NF 25 preps Gel Loading Buffer 50 g 100 g 50 g 100 g 50 g 100 g 50 g 100 g 50 g 100 g 1 ml 10 ml bags plates 20 plates 1 ml 5 x 1 ml 1 ml 5 x 1 ml Microbiological Culture Media Catalog No. Quantity TB DRY Powder Growth Media kg LB Broth Powder Growth Media, ph LB Agar Powder Growth Media, ph LB Broth (Lennox) Powder Growth Media, ph 7 LB Agar (Lennox) Powder Growth Media, ph 7 Soybean-Casein Digest Medium (TSB), USP Soybean-Casein Digest Agar Medium (TSA), USP Yeast Extract kg 5 kg 5 kg TAE Buffer, 50X (Tris-acetate-EDTA) TBE Buffer, 10X (Tris-borate-EDTA) RNase-Free Gloves 1556-XS 1556-S 1556-M 1556-L UltraClean Lab Cleaner kg KAPA PROBE FAST qpcr s kg 5 kg 5 kg KAPA SYBR FAST Universal 2X qpcr Master Mix KAPA2G Robust HotStart ReadyMix ml 1 liter 500 ml 1 liter bag of 150 bag of 150 bag of 150 bag of ml squeeze bottle 500 ml spray bottle 1 liter bottle 100 reactions 500 reactions 1000 reactions 100 reactions 500 reactions 1000 reactions 100 reactions 500 reactions Tryptone Agar, Bacteriological Grade kg 5 kg KAPA HiFi HotStart ReadyMix KAPA2G FAST HotStart DNA Polymerase with dntps reactions 500 reactions 100 reactions 250 reactions 500 reactions 17

18 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Other Reagents and Lab Accessories Continued Catalog No. Quantity KAPA2G FAST HotStart ReadyMix reactions reactions KAPA Long Range HotStart DNA Polymerase with dntps reactions 250 reactions 500 reactions Instrumentation and Accessories Continued Catalog No. Quantity Garnet Bead Tubes, 0.70 mm x 2 ml Garnet + ¼ Ceramic 15 ml Bead Tubes, 0.70 mm Garnet + ¼ Ceramic 50 ml Bead Tubes, 0.70 mm ` KAPA Taq Polymerase ReadyMix reactions Glass 15 ml Bead Tubes, 0.1 mm DNase (RNase-Free) Proteinase K Ribonuclease A (25 mg/ml) PCR Water Molecular Biology Grade Water DEPC Treated Water Endotoxin-Free Water UltraClean 5N NaOH UltraClean 0.1N NaOH UltraClean 0.5M EDTA, ph UltraClean TE-4 Buffer UltraClean TE Buffer UltraClean 1X PBS, ph 7.4 UltraClean 2M Tris, ph 8.0 UltraClean 0.1M Tris, ph mg 2500 units 100 mg 2 ml (20 mg/ml) 1 ml 5 ml 1 ml 5 x 1 ml 10 x 1 ml 10 ml bottle 200 ml 5 x 200 ml 200 ml 5 x 200 ml 10 ml 50 ml 100 ml 500 ml 30 ml 30 ml 100 m 1L 1L 500 ml 250 ml 1L Glass 50 ml Bead Tubes, 0.1 mm Glass 15 ml Bead Tubes, 1.0 mm Ceramic 15 ml Bead Tubes, 1.4 mm Ceramic 50 ml Bead Tubes, 1.4 mm Metal 50 ml Bead Tubes, 2.38 mm Ceramic Beads, 1.4 mm gm Ceramic Beads, 2.8 mm gm Glass Beads, 0.5 mm gm Metal Beads, 2.38 mm m Glass Beads, 0.1 mm gm Carbide Beads, 0.25 mm m Garnet Beads, 0.15 mm m Garnet Beads, 0.70 mm m Instrumentation and Accessories Catalog No. Quantity PowerMix 15 ml Bead Tubes PowerLyzer 24 Bench Top Bead- Based Homogenizer (110/220V) unit PowerMix 50 ml Bead Tubes PowerLyzer Tube Holder unit PowerLyzer Tube Holder Stand unit 2 ml Collection Tubes T PowerVac Mini System unit T adapters T PowerVac Manifold unit 2 ml Screw Cap Tubes E 200 & caps PowerVac Mini Spin Filter adapters 15 ml Collection Tubes T 25 Adapters adapters 50 ml Centrifuge Tubes T 25 Ceramic Bead Tubes, 1.4 mm bead Ceramic Bead Tubes, 2.8 mm bead Spin Filters (in 1.9 ml ) SF SF Glass Bead Tubes, 0.5 mm bead SF 50 filters 100 filters 250 filters Glass Bead Tubes, 0.1 mm bead Endotoxin-Free Centrifuge Tubes each/2 ml Metal Bead Tubes, 2.38 mm bead each/15 ml 2.0 ml Tough Tubes with Cap each/50 ml Carbide Bead Tubes, 0.25 mm x 0.5 ml Garnet Bead Tubes, 0.15 mm x 0.5 ml 15 ml Midi Spin Filters SF 25 spin filters

19 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Instrumentation and Accessories Continued Catalog No. Quantity Vortex-Genie 2 Vortex (120V) V 1 unit Vortex-Genie 2 Vortex (220V) V unit Vortex Adapter, holds 12 ( ml) V1 1 unit Vortex Adapter, holds 6 (5 ml) V1-5 1 unit Vortex Adapter, holds 4 (15 ml) V unit Vortex Adapter, holds 2 (50 ml) V unit Vortex Adapter, holds 24 ( ml) V unit Anti-Static Funnels, Micro Pack of 96 Anti-Static Funnels, Small Pack of 50 Anti-Static Funnels, Medium Pack of 50 Anti-Static Funnels, Large Pack of 20 Mini Horizontal Gel System each Mini Horizontal Gel Caster, 3 place each Mini Horizontal Gel Tray each Polycarbonate Single-sided Comb Polycarbonate Dual-sided Comb Teflon Single-sided Comb Teflon Dual-sided Comb Power Supply w/timer, (120V) unit 96 Well Plate Shaker (120V) unit 1 mm x 3 well 1 mm x 8 well 1 mm x 10 well 1 mm x 12 well 1 mm x 8 well/16 well 1 mm x 10 well/14 well 2 mm x 8 well/16 well 2 mm x 10 well/14 well 1 mm x 3 well 1 mm x 8 well 1 mm x 10 well 1 mm x 12 well 1 mm x 8 well/16 well 1 mm x 10 well/14 well 2 mm x 8 well/16 well 2 mm x 10 well/14 well 96 Well Plate Shaker (220V) unit Plate Adapter Set set Vacuum Pump (120V) unit Vacuum Pump (220V) unit 19