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User Manual and Troubleshooting

1. KR RR RR RR RR RR RR KR RR RR KR RR RR RR RR RR RR RR KR RK Address NeuBiogene Inc 2275 East Foothill Blvd Pasadena CA 91107 Tel 626 927 8477 For technical support email tech_support neubiogene com NeuBiogene Inc sor www neubiogene com sooo Fax 626 577 8489 Troubleshootings 1 Weak hybridization signals following reasons may contribute to the weak signals Hybridization temperature is not optimized Suggestion change the hybridization temperature based on the size of the probe Use 60 C for gt 100nt probe and 50 C 50 100nt probes Lower quality of the probes e Probes are hydrolyzed after synthesis or probes e The radioactivity of labeled probes is too low less then 10 cpm ul e Probes added into the hybridization solution are not enough for maximal hybridization Suggestion the radioactivity of isotopic probes in the hyb solution should be 10 cpm ml and for the no isotopic probe it should be 100ng ml The abundance of target genes are low the most common reason for weak signals Suggestion increase the amounts of target genes on hybridization membrane 2 High background kk RR KR KK Parts of the membrane are dried out during hybridization step or washing step so that the probes adhere to the membrane Labeled free nucleotides are not completely removed from the probes Suggestion after generating probes by in vitro transcription use NeuBiogene RNA cleaning up kit to remove labeled free nucleo
2. NeuBiogene Inc roe www neubiogene com mooo Fax 626 577 8489 User Manual Read all the procedures before using Super Hybridization Buffer for experiment User Manual for membrane hybridization only 1 Thaw the Superhyb solution at 60 C Repeat freezing and thaw is OK 2 Shake the solution to mix it well and add solution to membrane in the hybridization tube or bag For 10cm x10 cm membrane use 10 ml of SuperHyb solution 3 Move hybridization bag or tube so that the solution can cover all surface of the membrane 4 After prehyb for 30 min at 60 C add certain amount probes 100ng ml for non isotopic probes and 1x10 cpm ml for isotopic probes directly to the hybridization tube or bag without changing hybridization solution 5 Hybridization is performed at 60 C for gt 50nt RNA probes or at 50 50 C for gt 50b DNA probes 2 8h hyb is sufficient for most abundant genes Overnight hyb is necessary for low abundant genes such as transcription factors receptor genes etc 6 After hybridization wash membrane with 1XSSC at hybridization temperature for 2X10 min following by wash in 0 2XSSC for 2X10 min at 5 10 C below the hybridization temperature 7 Air dry membrane after washes and detect the hybridization signals by exposure to film or image box isotopic labeled probes or by immunocytochemistry non isotopic labeled probes KR RR KR RR RR RR RR RR RR RR RR KR RR RR RR RR RR RR RR RR RR RRR RK RR RR RR RR RR RR RR RR
3. tides from the probes KR KR RRR RR KR KR KR RR RR RR RR RR RR RR RR KR RR RR RR RR RR RR RR RR RR RR RRR RR RR RR RR RR RR RR RR RR KR RR RR RR RR RR RR RR RR RR KK Address NeuBiogene Inc 2275 East Foothill Blvd Pasadena CA 91107 Tel 626 927 8477 For technical support email tech_support neubiogene com

User Manual and Troubleshooting

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