PerfectHyb hybridization solution
Contents
1. 60 40 Relative signal intensity iJ Oo 0 2 4 6 8 10 12 14 16 18 Hybridization time hours Fig 2 Relationship between hybridization time and relative signal intensity when detecting a rarely expressed mRNA transferrin receptor by Northern blot 4 Com po nents This reagent contains the following components PerfectHyb hybridization solution 250 ml This product can be stored at room temperature In the case of long term storage this product should be stored at 2 8 C At lt 15 C a precipitate may form If this occurs the solution should be heated to 37 C and thoroughly mixed to completely dissolve precipitate 5 Materials required The following materials are required but not supplied 1 Reagents Wash solution A 2x SSC pH 7 0 0 1 SDS Wash solution B 0 1x SSC pH 7 0 0 1 SDS This reagent is required for Southern blots with all probes as well as Northern blots using a RNA probe see 7 Reagent 2 Instruments Heating bath with shaker or hybridization oven Heat sealer when using hybridization bag Hybridization bag optional X ray film optional JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 2 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE www toyobo co jp e bio 6 Protocol2. TOYOBO Bio Technology CO LTD Tel 86 21 58794900 4140 FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE www toyobo co jp e bio 7 Reagents 1 20x SSC 3 M NaCl 0 3 M Sodium citrate 1000 ml 175 g NaCl 88 g tri sodium citrate dihydrate Dissolve in 900 ml distilled water Adjust pH to 7 0 with 1N HCl Adjust volume to 1000 ml with distilled water Store at room temperature 2 10 SDS 500 ml 50 g SDS Adjust volume to 500 ml with distilled water Store at room temperature 3 Wash solution A 2x SSC 0 1 SDS 500 ml 50 ml 20x SSC 5 ml 10 SDS Adjust volume to 500 ml with distilled water Store at room temperature 4 Wash solution B 0 1x SSC 0 1 SDS 500 ml 2 5 ml 20x SSC 5 ml 10 SDS Adjust volume to 500 ml with distilled water Store at room temperature 5 20 x SSPE 3 M NaCl 173 mM sodium dihydrogen phosphate 25 mM EDTA 1000 ml 175 g NaCl 27 g sodium dihydrogen phosphate dihydrate 7 4 g EDTA 2Na Dissolve in 800 ml distilled water Adjust pH to 7 4 with 5 N NaOH Adjust volume to 1000 ml with distilled water Store at room temperature JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 7 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio 8 Related protocol 1 Stripping of membranes The following protocol is a typical stripping method fo
3. 6 1 Northern and Southern blot analyses using cDNA and cRNA probes 1 Preparation of probes Probe labeling should be performed according to the instruction manual The free nucleotides should be removed prior to use NOTES The specific activity of radioactive probes should be confirmed prior to use The radioactive probes should be labeled just before use Non radioactive probes should be confirmed by a spot test in accordance with the instruction manual Probes with direct repeats or long probes 4 kb tend to generate unexpected extra bands or background signals Probe sequences should be confirmed prior to preparation 2 Hybridization and washing conditions The following table provides hybridization and washing conditions Probe Label Hybridization Wash Wash Solution 1 Solution 2 Time 1h 5 minutes 15 minutes overnight x2 x2 Northern DNA RI 68 C 68 C A 68 C A Blot Non RI RNA Non RI 68 C 68 C A 68 C B Southern DNA RI 68 C 68 C A 68 C B Blot Non RI A Wash solution A B Wash solution B NOTES When using a heating bath for hybridization hybridization should be performed in a hybridization bag The membranes should be washed with shaking using a container A hybridization oven is suitable for the analyses with radioactive probes In this case membranes should be washed in the hybridization oven 3 Probe concentration The following table lists recommended probe concentrations Higher
4. cDNA cRNA and oligonucleotide probes can be used for Northern blot analysis Radioactive cDNA or oligonucleotide probes are recommended for analyses using commercially available pre blotted membranes mRNA blotted membranes Please read this instruction manual prior to use to prevent unexpected background signals in particular with non radioactive methods This reagent has been evaluated using commercially available pre blotted membranes Southern blot Radioactive as well as non radioactive DNA and oligonucleotide probes are recommended for Southern blot analysis Please read this instruction manual prior to use to prevent unexpected background signals in particular with non radioactive methods 3 Princi p 7 This reagent reduces hybridization time due to a rate enhancer included in the solution On Northern blots this reagent also enhances signal intensity 120 a 100 S 8 80 3 O Northern 2 a Southern 2 z 0 0 2 4 6 8 10 12 14 16 18 Hybridization time hours Fig 1 Relationship between hybridization time and relative signal intensity on Northern and Southern blots using DIG labeled B actin and VNTR probes JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 1 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio PerfectHyb OConventional
5. concentrated probes tend to generate background signals in particular when employing non radioactive probes Label Probe Probe concentration RI DNA 1 2 x 10 cpm ml or 1 10 ng ml Non RI DNA 0 2 1 ng ml RNA JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 3 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio 4 Hybridization time The following table lists hybridization times Expression level of Hybridization time target mMRNA Southern blot 1 hour Northern blot High 1 hour Medium 2 hours Low 2 hours overnight When using long probes gt 4 kb Overnight Long probes gt 4 kb tend to generate background signals Overnight hybridization reduces background levels when using the long probes 4 kb Probe concentration should be reduced on analyses with long 4 kb non radioactive probes NOTES Probe concentration and hybridization time should be determined according to the probe concentration and expression levels of the target genes Overnight hybridization with a radioactive probe is recommended for detection of mRNA with low or unknown expression levels 5 Protocol a Prehybridize 10 x 10 cm 100 cm membranes in a minimum volume of 5 ml PerfectHyb hybridization solution at 68 C for 20 minutes b Denature labeled
6. probe at 100 C for 5 minutes Labeled probes should be denatured in low salt water e g distilled water or TE buffer just prior to use RNA probes should be denatured prior to use c In the case of 100 cm membranes add the denatured probe to a minimum volume of 5 ml pre warmed fresh PerfectHyb hybridization solution Assure that probe is thoroughly mixed with fresh hybridization solution d Replace pre hybridization solution with fresh hybridization solution containing the labeled probe e Incubate at 68 C for 1 hour overnight Wash membrane two times in pre warmed Wash solution 1 at 68 C for 5 minutes g Wash membrane two times in pre warmed Wash solution 2 at 68 C for 15 minutes h Radioactive probe Transfer membrane with forceps onto filter paper Then follow with exposure to X ray film at 70 C Non radioactive probe Perform detection according to the instruction manual JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 4 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio 2 Analysis using oligonucleotide probes 1 Preparation of probes Probes should be labeled according to instruction manual The free nucleotides should be removed prior to use Probe labeling efficiency should be checked prior to use 2 Calculation of Tm val
7. using non radioactive probes the detection should be performed according to the instruction manual Direct repeats e g Alu in the probe sequence generate extra bands Additional bands may be reduced by including denatured salmon sperm DNA final concentration 100 ug ml Insufficient washing Increasing washing steps or decreasing SSC concentration e g 1x SSC gt 0 5x SSC may eliminate the extra bands 10 Related products Product name Package Code No MagExtractor PCR amp Gel Clean up 1 kit NPK 601 Magnetic stand 1 piece MGS 101 Magical Trapper JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 10 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE
8. Northern blot Reprobing Reprobing decreases signal intensity Prolong the exposure time Too much washing Reduce the washing time Increasing SSC concentration e g 0 1x SSC gt 0 2x SSC may enhance the signal Blotted nucleic acids were not sufficient Increase the amount of nucleic acids on the membrane Extra bands Too much labeled free nucleic acid Remove the labeled free nucleic acids prior to use Probe concentration was too high Decrease probe concentration High concentration probes tend to generate high background signals Length or sequence of the probe was not appropriate Probes prepared from a long template gt 4 kb or possessing repeat sequences tend to generate non specific signals Reconfirm the template size and sequence High background signals Drying of the membrane at hybridization steps Drying of the membrane at the hybridization step increases background signals Take care that the membrane does not become dry Foaming of the hybridization solution in the hybridization bag Foaming of the hybridization solution causes irregular spot signals Remove bubbles from the hybridization bag prior to hybridization Insufficient washing solution Use sufficient volumes of washing solution Detection method was not inappropriate The probe contained non specific sequences Non radioactive probes tend to generate high background signals When
9. OBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 8 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio 2 Purification of radioactive probes The following protocol is a simple purification method for radioactive cDNA probes using a DNA fragment purification kit e g MagExtractor PCR amp Gel clean up 1 Materials required MagExtractor PCR amp Gel clean up Code No NPK 601 75 Ethanol Magnetic stand e g Magical trapper Code No MGS 101 2 Protocol a Dispense lt 50 ul of labeled DNA probe into a 1 5 ml microtube b Add 200 ul Binding Solution c Binding Add 15 ul Magnetic Beads and vortex the tube every 10 seconds for 1 2 minutes d Place the tube in the magnetic stand The magnet will attract the magnetic beads separating then from the specimen solution Notes ae Completely resuspend the magnetic beads prior to use P me Fig 1 e Upon magnetic capture carefully remove the supernatant Magnetic separation Washing Add 300 ul Washing Solution to the beads and vortex for 10 seconds g Place the tube in the magnetic stand and collect the beads with the magnet h Upon magnetic capture carefully remove the supernatant and discard into a waste tank i Washing Add 1 ml 75 EtOH to the tube and vortex for 10 seconds j Place the tube in the magnetic stand and col
10. e bio 5 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO JAPAN TOYOBO CO LTD Tel 81 6 6348 3888 www toyobo co jp e bio tech_osaka toyobo jp www toyobo co jp e bio 4 Probe concentration The following table lists recommended probe concentrations Higher concentrated probes tend to generate background signals in particular with analyses employing non radioactive probes Label Probe concentration RI 2 5 5 pmoles ml Non RI 0 5 1 pmoles ml 5 Protocol a Prehybridize 10 x 10 cm membranes in a minimum total volume of 5 ml of PerfectHyb hybridization solution at optimal hybridization temperature for 20 minutes b Add the labeled probe to 5 ml of prewarmed fresh PerfectHyb hybridization solution Assure that probe is thoroughly mixed with fresh hybridization solution c Replace pre hybridization solution with fresh hybridization solution containing the labeled probe e Incubate at optimal hybridization temperature for 1 2 hours f Wash membrane two times in pre warmed Wash solution 1 at the optimal temperature for 5 minutes g Wash membrane two times in pre warmed Wash solution 2 at the optimal temperature for 10 minutes h Radioactive probe Transfer membrane with forceps to filter paper Then follow with exposure to X ray film at 70 C Non radioactive probe Perform detection according to the instruction manual CHINA
11. lect the beads with the magnet k Upon magnetic capture carefully remove the supernatant Notes The 75 EtOH should be completely removed after flash centrifugation 1 lt Elution gt Add 25 50 ul sterilized water and mix well for 10 seconds m Incubate at room temperature for 2 minutes n Place the tube in the magnetic stand after briefly vortexing o Collect the supernatant and place into a fresh tube Notes Handling should be performed opposite an acrylic board 1 5 ml microtubes with screw caps should be used to prevent contamination JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio 9 tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE 9 Troubleshooting www toyobo co jp e bio absent or very week denatured Symptom Cause Solution Insufficient exposure Prolong exposure time Specific activity or labeling Confirm specific activity or labeling efficiency of efficiency of probe was too probes low Probe concentration was too Increase probe concentration low Labeled probe was not fresh When using radioactive probes the probe should be labeled just prior to use Do not store the probe Hybridization signals Labeled probe was not Denature the probes just prior to use Insufficient hybridization time Overnight hybridization is effective for detecting low expressing mRNA on
12. r membranes following analysis with radioactive probes NOTES Membrane stripping tends to decrease signals with the subsequent blot Therefore the membrane should be baked followed by UV irradiation cross linking prior to re use When using commercially available pre blotted membranes mRNA blotted membranes the membrane should be stripped according to the corresponding instruction manual If the membrane has an indication concerning the stripping method stripping should be performed according to that indication Stripping efficiency may depend on the types of probes 1 Materials required a Stripping reagent 100 ml 55 ml formamide 10 ml 20x SSPE 5 ml 10 SDS Adjust volume to 100 ml with distilled water This solution should be prepared just prior to use b Wash solution B 0 1x SSC pH 7 0 0 1 SDS gt see 7 reagent c Hybridization oven or heating bath 2 Protocol a Transfer the membrane to a hybridization bath or bottle b Add 10 ml of stripping solution to the 10 cm x 10 cm 100 cm membrane c Incubate the membrane at 68 C for 1 2 hours d Discard stripping solution and wash membrane at 68 C for 10 minutes Notes The radioactivity of stripped membranes should be measured with a survey counter or X ray film When using X ray film exposure should be performed overnight Upon stripping the membrane should be stored properly to prevent drying JAPAN CHINA TOYOBO CO LTD TOY
13. ue of probes The Tm value of each probe should be determined according to the following equation a Oligonucleotide probe lt 18 base Tm A T x 2 C G C x 4 C b Oligonucleotide probe 18 base Tm 81 5 16 6 log 10 Na 0 41 G C 600 N A the number of adenine bases in the oligonucleotide T the number of thymine bases in the oligonucleotide G the number of guanine bases in the oligonucleotide C the number of cytosine bases in the oligonucleotide G C percentage of G C in the oligonucleotide N the number of nucleotides in the oligonucleotide Na 0 75 M 3 Hybridization and washing conditions The following table lists hybridization and washing conditions Hybridization Wash Solution 1 Wash Solution 2 Time 1 2 hours 5 minutes x 2 10 minutes x 2 Radioactive probe ane Non radioactive probe A TWEN TA A Wash solution A NOTES When using probes with a high Tm 70 C hybridization should be performed at 55 60 C When using a heating bath for hybridization hybridization should be performed in a hybridization bag The membranes should be washed with shaking using a container A hybridization oven is suitable for analyses with radioactive probes In this case the membrane should be washed in the hybridization oven JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp
14. www toyobo co jp e bio F1017K PerfectHyb hybridization solution HYB 101 250ml Store at room temperature Contents 1 Introduction 2 Applications 3 Principle 4 Components 5 Materials required 6 Protocol 1 Northern and Southern blot analysis using cDNA and cRNA probes 2 Analysis using oligonucleotide probes 7 Reagents 8 Related protocols 9 Troubleshooting 10 Related products CAUTION All reagents in this kit are intended for research purposes Do not use for diagnostic or clinical purposes Please observe general laboratory precautions and safety measures while using this kit JAPAN CHINA TOYOBO CO LTD TOYOBO Bio Technology CO LTD Tel 81 6 6348 3888 Tel 86 21 58794900 4140 www toyobo co jp e bio tech_osaka toyobo jp FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE TOYOBO www toyobo co jp e bio 1 Introduction pecerigtien PerfectHyb is an easy to use hybridization solution which contains a rate enhancer This reagent has been optimized for Northern and Southern blot analyses and includes the following features Features Reduced hybridization time from the customary 12 24 hours to 1 2 hours Allows for the use of radioactive and non radioactive nucleic acid probes Enhanced signals using Northern blot analysis Same temperature for hybridization and washing steps No requirement for salmon sperm DNA Low viscosity allows for easy handling 2 Applications Northern blot
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