新闻动态
The Neverfail Northern Blot Hybridizatio - 实验方法
2025-06-17
相关专题 PreHybridization/Hybridization Solution
0.1% SDS
50% Formamide
5x SSC
50 mM NaPO4, pH 6.8
0.1% Sodium Pyrophosphate
5x Denhardt s Solution
50 ug/ml Sheared Herring Sperm DNA
1) Air dry blot. Crosslink in Stratalinker or 30 seconds on UV Transillumina tor. Wash the blot for 30 minutes in 1x SSC, 0.1% SDS at 65℃.(optional)
2) Drain solution and replace with prehyb solution. Prehyb for at least 2 hours at 42℃.
3) DeNature the probe at 95℃ for 5 minutes.
4) Replace prehyb solution with fresh, preheated hyb solution containing 1 million cpm of probe per ml of hyb solution. (Average hyb is 10 million cpm total).
5) Hybridize overnight at 42℃.
6) Wash # 1: Fresh prehyb solution, 30 minutes at RT.
7) Wash #2 and #3: 2x SSC, 0.1%SDS 30 minutes each at RT.
8) Wash #4: 1x SSC, 0.1% SDS for 30 minutes at RT.
9) Wash #5: 0.2x SSC, 0.1% SDS for 45 minutes at 55℃.
10) Blot membrane dry with Kimwipe, cover with Saran Wrap and expose to film.
Troubleshooting and TiPS Excessive Background on film: Speckled background indicates that there are unincorporated nucleotides in the probe.
Blotchy backround indicates non-specific hybridization. This can be caused by salt still on the blot before the hyb. Be sure to wash the membrane. Can also indicate a lack of agitation during the hyb. No signal: a) Probe targets intronic sequences.
b) Probe not deNature d prior to hyb.
c) Hyb conditions too stringent.
d) Washing conditions too stringent. Hybridization of Ribosomal RNA: a) Too much total RNA has been blotted. Do not exceed 20ug of total RNA or try to use poly A+ instead.
b) Probe is too sticky. Increase the stringency of the hyb conditions. Smears: The RNA has degraded. Stain the gel with Ethidium Bromide or SYBR Green II to ensure the RNA is intact. Can also stain filter with Methylene Blue to ensure the RNA did not degrade during transfer process. Stripping blots for reprobing: Add boiling 0.1% SDS, and wash for 30 minutes. Reprobe starting with step 2.
您知悉并同意，丁香通平台展示产品并非为平台自身产品，您发起询价或试用申请后，平台将会把您已提供或按要求提供的相关信息同步提供至所咨询的具体产品商家，由商家为您提供具体产品的价格咨询或产品试用注意事项及相关试用产品或完成相应购买，因此您知悉并授权平台将您的信息进行同步共享。您有权拒绝，但您知悉将无法参加询价或试用活动。
0.1% SDS
50% Formamide
5x SSC
50 mM NaPO4, pH 6.8
0.1% Sodium Pyrophosphate
5x Denhardt s Solution
50 ug/ml Sheared Herring Sperm DNA
1) Air dry blot. Crosslink in Stratalinker or 30 seconds on UV Transillumina tor. Wash the blot for 30 minutes in 1x SSC, 0.1% SDS at 65℃.(optional)
2) Drain solution and replace with prehyb solution. Prehyb for at least 2 hours at 42℃.
3) DeNature the probe at 95℃ for 5 minutes.
4) Replace prehyb solution with fresh, preheated hyb solution containing 1 million cpm of probe per ml of hyb solution. (Average hyb is 10 million cpm total).
5) Hybridize overnight at 42℃.
6) Wash # 1: Fresh prehyb solution, 30 minutes at RT.
7) Wash #2 and #3: 2x SSC, 0.1%SDS 30 minutes each at RT.
8) Wash #4: 1x SSC, 0.1% SDS for 30 minutes at RT.
9) Wash #5: 0.2x SSC, 0.1% SDS for 45 minutes at 55℃.
10) Blot membrane dry with Kimwipe, cover with Saran Wrap and expose to film.
Troubleshooting and TiPS Excessive Background on film: Speckled background indicates that there are unincorporated nucleotides in the probe.
Blotchy backround indicates non-specific hybridization. This can be caused by salt still on the blot before the hyb. Be sure to wash the membrane. Can also indicate a lack of agitation during the hyb. No signal: a) Probe targets intronic sequences.
b) Probe not deNature d prior to hyb.
c) Hyb conditions too stringent.
d) Washing conditions too stringent. Hybridization of Ribosomal RNA: a) Too much total RNA has been blotted. Do not exceed 20ug of total RNA or try to use poly A+ instead.
b) Probe is too sticky. Increase the stringency of the hyb conditions. Smears: The RNA has degraded. Stain the gel with Ethidium Bromide or SYBR Green II to ensure the RNA is intact. Can also stain filter with Methylene Blue to ensure the RNA did not degrade during transfer process. Stripping blots for reprobing: Add boiling 0.1% SDS, and wash for 30 minutes. Reprobe starting with step 2.
您知悉并同意，丁香通平台展示产品并非为平台自身产品，您发起询价或试用申请后，平台将会把您已提供或按要求提供的相关信息同步提供至所咨询的具体产品商家，由商家为您提供具体产品的价格咨询或产品试用注意事项及相关试用产品或完成相应购买，因此您知悉并授权平台将您的信息进行同步共享。您有权拒绝，但您知悉将无法参加询价或试用活动。
本文链接： https://www.ebiomall.cn/b535-hybrid/info-12953.html
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
相关文章
2020-07-06
2020-07-19
2020-09-07
2020-09-17
2020-09-26
2020-09-28
2020-10-12
▍
官网分类
▍
品牌问答
