Bio-rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit Manual de usuario

Aurum™Total RNA Fatty andFibrous Tissue Kit Instruction ManualCatalog # 732-6830The Aurum Total RNA Fatty and Fibrous Tissue Kit is composed of:Module

6yeast for processing. To calculate the volume of culture required, use the followingequation:(OD600of undiluted culture) x (culture volume in ml) = #

7ganerpinReagents Used With the Aurum Total RNA Fatty and FibrousTissue Kit PureZOL™ RNA Isolation Reagent for Sample Lysis Use 1 ml of PureZOL for up

8(DEPC) to inactivate RNases. DEPC is an efficient, strong, and nonspecificRNase inhibitor that is usually used at a concentration of 0.1%• To prepare

9• Nondisposable, nonautoclavable plasticware should be rinsed with 0.1 MNaOH, 1 mM EDTA followed by several rinses with DEPC-treated water beforeuse•

10Table 3. Disruption and homogenization methods.StartingMaterial Disruption Method Homogenization MethodAnimal tissue Grind tissue with a Pipetting u

11Section 6Vacuum Manifold Setup and Use With theColumn Adaptor PlateGuidelines for Vacuum Format• The recommended operating range is –17 to –20 inHg.

12Vacuum Setup (Figure 1)1. Cut tubing into three pieces of appropriate length.2. Use one piece of tubing to connect to the right side of the vacuumr

13Manifold Wash Setup (Figure 2)1. Insert the CAP (luer ends up) into the depression in the vacuum manifoldtop. Ensure that the CAP rests evenly on t

14Section 7Vacuum ProtocolAll steps are carried out at room temperature unless otherwise indicated.Vacuum filtration steps should be carried out at –1

15Warning: Processing larger amounts of starting material may lead to columnclogging and reduced RNA purity. It is crucial that the appropriate amount

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Cells Grown in a Monolayer Cells grown in a monolayer should be lysed with PureZOL directly in theculture dish. Aspirate the culture medium and immedi

174. Add 0.2 ml of chloroform to the lysate, then cover and shakevigorously for 15 sec. Do not vortex!5. Incubate for 5 min at room temperature while

10. Pipet 700 µl of the RNA sample into the RNA binding minicolumn. Turn the vacuum on and adjust to –17 to –23 inHg by closingthe vacuum regulator. C

1918. Pipet 40 µl (or 30 µl)†of the elution solution onto the center ofthe membrane at the bottom of the RNA binding column. †Note: When isolating tot

Section 8Spin ProtocolImportant: If using the kit for the first time, please read Section 6, "BeforeUsing the Aurum™ Total RNA Fatty and Fibrous

21up to 100 mg of freshly dissected tissue into a 2.0 ml microcentrifuge tubeand add 1 ml of PureZOL. Disrupt the sample for 30–60 sec using a rotor-s

Note: Do not wash cells prior to the addition of PureZOL as this couldincrease the possibility of mRNA degradation. 3. Once the sample has been disrup

23b. For each column to be processed, mix 5 µl of reconstituted DNase I with75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube. Scale u

14.Add 700 µl of high stringency wash solution to the RNA bindingcolumn. Centrifuge for 30 sec at >12,000 x g. Discard the highstringency wash solu

Section 9Troubleshooting GuideProblems that may be encountered during RNA purification:Problem Possible Cause Recommended SolutionIncomplete Lysate wa

Table of ContentsSection 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Section 2 Kit Components . . . . . . . . . . . . .

Problem Possible Cause Recommended SolutionRNA binding mini Starting material is After the sample column is clogging high in fat, proteins, disruptio

Problem Possible Cause Recommended SolutionLow RNA yield Low amount of Do not use less than the(continued) starting material recommended minimumstarti

Problem Possible Cause Recommended SolutionGenomic DNA On-column DNase I Perform the on-columncontamination digest was not DNase I digest (see steppe

Problem Possible Cause Recommended SolutionRNA is degraded Frozen tissue samples Add PureZOL directly to(continued) were allowed to thaw or frozen s

Problem Possible Cause Recommended SolutionLow RNA A260/A280Some of the white Leave some of theratio interphase and the aqueous phase solution(continu

Problem Possible Cause Recommended SolutionPrepared total RNA RNA is degraded See troubleshootingperforms poorly section "RNA isin downstream de

Section 10Ordering InformationCatalog # Description732-6830 Aurum Total RNA Fatty and Fibrous Tissue Kit732-6870 Aurum Total RNA Fatty and Fibrous Ti

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Bio-Rad Laboratories, Inc.2000 Alfred Nobel Dr.Hercules, CA 94547 USA(510) 741-10001-800-424-672310001298 Rev B10001298B:4110133A.qxd 1/16/2009 2:

Section 1Introduction The Aurum™ total RNA fatty and fibrous tissue kit produces high yields ofpure total RNA from samples that are difficult to disru

Section 2Kit ComponentsThe Aurum™ total RNA fatty and fibrous tissue kit contains the followingcomponents:Components* Quantity/AmountRNA binding mini

Section 3Storage ConditionsStore components of the kit at the recommended temperatures (see Table 1below).Table 1. Recommended storage temperature for

Supplies for Tissue Grinding, Disruption, and Homogenization• Fresh tissue: tissue cutter• Frozen tissue: liquid nitrogen, mortar and pestle• Tissue h

5Section 5Before Using the Aurum™ Total RNAFatty and Fibrous Tissue KitPlease read the following guidelines before proceeding with the total RNAisolat