TECHNICAL MANUAL ReliaPrep™ Large Volume HT gDNA Isolation System Instruc ons for use of Products A1751 AND A2751 For Research Use Only. Not for use in diagnos c procedures. TM341 Revised 7/13

ReliaPrep™ Large Volume HT gDNA Isolation System All technical literature is available on the Internet at: www.promega.com/protocols/ Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail: [email protected] The ReliaPrep™ Large Volume HT gDNA Isolation System isolates genomic DNA (gDNA) from blood samples ranging from 1ml to 10ml of blood in a scalable format. The chemistry eliminates tedious centrifugation steps as well as the use of hazardous chemicals,...

Sample processing is simplified through the use of the HSM 2.0 Instrument (Cat.# A2715), which heats, shakes and magnetizes sample tubes in one position throughout the extraction process from lysis to elution. This system removes resource limitations from sample processing and increases automated method robustness and reliability. Genomic DNA yields from normal 10ml whole blood samples are typically 200-400(rg (depending on white blood cell count) in a final eluted volume of 1ml. Recovered DNA exhibits good purity with A260/A28o ratios greater than 1.7 and A260/ A230 ratios between 1.8 and 2.2....

Product Components and Storage Conditions Product Cat.# ReliaPrep™ Large Volume HT gDNA Isolation System A1751 Each system contains sufficient reagents for 96 isolations from up to 10ml each or 320 × 1–3ml in a scalable format. Includes: • 23ml • 130ml • 1,020ml • 2 × 765ml • 115ml • 3 × 815ml • 2 ×150ml Proteinase K (PK) Solution Alkaline Protease (APA) Cell Lysis Buffer (CLD) Binding Buffer (BBA) ReliaPrep™ Resin Wash Buffer (WBA) Nuclease-Free Water Product Cat.# ReliaPrep™ Large Volume HT gDNA Isolation System A2751 Each system contains sufficient reagents for 96 isolations from up to 10ml...

General Considerations 3.A. Comparison with Organic- and Precipitation-Based Methods The ReliaPrep™ Large Volume HT gDNA Isolation System addresses the challenges of traditional genomic DNA purification methods that rely on centrifugation-based organic or precipitation chemistries by isolating genomic DNA from the entire lysed whole blood sample. The ReliaPrep™ Large Volume HT gDNA Isolation System efficiently purifies genomic DNA regardless of whether blood samples are fresh or frozen. 3.B. Sample Processing Capacity Genomic DNA yield depends on 1) volume of whole blood processed and 2) number...

3.E. Manual Versus Automated Processing For more information about the HSM 2.0 Instrument, see the HSM 2.0 Instrument Technical Manual #TM389 at: www.promega.com/protocols/ Manual Processing The HSM 2.0 Instrument is controlled by software installed on a computer with the Windows® 7 operating system with methods to process 1–10ml of blood. These methods guide the user through the purification process with specific instructions on reagent addition and waste removal. Solutions are typically added with a pipette and aspirated using a vacuum aspirator. Automated Processing Automated processing is...

Materials to Be Supplied by the User • HSM 2.0 Instrument (Cat.# A2715) with HSM 2.0 Tube Rack, HSM 2.0 Tube Rack Stand and computer with HSM 2.0 Instrument software installed (see HSM 2.0 Instrument Operating Manual #TM389 for details) 47.5–50% ethanol, at room temperature, prepared using Molecular Biologygrade water (see Section 4.A) 50ml conical tubes (one per sample, e.g., Corning Cat.# 430291) 10ml pipettes and pipette aid Optional: A repeater pipette (Gilson or Eppendorf) for adding reagents to larger numbers of samples Be sure to remove all lysate, Prepared Wash Buffer and ethanol from...

5. Add the volume of Proteinase K Solution displayed onscreen to each tube. 6. Optional: Add the indicated volume of RNase A indicated on screen to each tube. 7. Select “OK” to start shaking samples at 500rpm for 1 minute. After the beep, add the volume of Alkaline Protease indicated on screen to each tube. Select “OK” to start shaking samples at 500rpm for 1 minute. 8. Add the appropriate volume of Lysis Buffer to each sample. After Lysis Buffer addition, the HSM 2.0 Instrument incubates the samples at 65°C for 30 minutes with shaking at 500rpm, followed by 10 minutes of shaking at 500rpm without...

4.B. Manual DNA Purification (continued) 15. The shaker will stop, and the HSM 2.0 Instrument collects the ReliaPrep™ Resin at the side of the tube for 3 minutes. Slowly aspirate the fluid from each tube when prompted by the instrument. Remove all Wash Buffer before adding ethanol in Step 16. After the initial removal, perform an additional aspiration to ensure that all Wash Buffer is removed. 16. Add the amount of 47.5–50% ethanol shown on screen to each sample. Select “OK” to start samples shaking samples at 500rpm for 4 minutes. 17. The shaker will stop, and the HSM 2.0 Instrument collects...

Quantitation and Analysis of Isolated Genomic DNA DNA quality and concentration can be determined by a variety of means including spectrophotometry, dye fluorescence, gel electrophoresis, and quantitative PCR. Different DNA quantitation methods frequently return dissimilar absolute quantitation values. We recommend that users select analysis methods for DNA quality and quantity that best predict success for the intended downstream applications. A260/A280 ratio can be used to determine DNA purity (with a number of important limitations [1–3]). An A260/A280 ratio between 1.7 and 2.0 is generally...

Troubleshooting (continued) Sample size exceeded the processing capacity of the system. The ReliaPrep™ Large Volume HT gDNA Isolation System is optimized to purify DNA from 1–10ml of whole blood. Processing blood samples that are outside of this range will result in reduced DNA yield and concentration. Exceeding the 10ml limit will cause the required processing volumes to exceed the maximum volume of a 50ml tube. The ReliaPrep™ Large Volume HT gDNA Isolation System is optimized for whole blood samples that contain between 4.5 x 106 and 1 x 107 white cells/ml. Whole blood samples containing more...