Chromosomal FISH-TSA: Difference between revisions
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Following protocol is designed for the visualization of short cDNA sequences with the minimal lenght of 900-1000 bp. | Following protocol is designed for the visualization of short cDNA sequences with the minimal lenght of 900-1000 bp. | ||
''' | '''Protocol:''' | ||
'''A. Preparation of labeled cDNA probe''' | '''A. Preparation of labeled cDNA probe''' | ||
We always use the cDNA probe of approx. 1000 bp in length depending on type of cDNA. Any | We always use the cDNA probe of approx. 1000 bp in length depending on type of cDNA. Any repete sequences in the probe could make a strong background using TSA. | ||
- prepare at least 2 μg of amplificate of cDNA from reverse transcription reaction by PCR and specific primers | - prepare at least 2 μg of amplificate of cDNA from reverse transcription reaction by PCR and specific primers | ||
Revision as of 11:50, 3 January 2011
Literature:
Krylov V., Macha J., Tlapakova T., Takac M., Jonak J. (2003) The c-SRC1 gene visualized by in situ hybridization on Xenopus laevis chromosomes. Cytogenet. Genome Res. 2003;103(1-2):169-172. [1].
Tlapakova T., Krylov V., Macha J. (2005) Localization, structure and polymorphism of two paralogous Xenopus laevis mitochondrial malate dehydrogenase genes. Chromosome res. 13(7):699-706. [2].
Krylov V., Tlapakova T., Macha J. (2007) Localization of Mdh2 single copy gene on Xenopus tropicalis chromosomes by FISH-TSA technique. Cytogenet. Genome Res. 116:110-112. [3].
Following protocol is designed for the visualization of short cDNA sequences with the minimal lenght of 900-1000 bp.
Protocol:
A. Preparation of labeled cDNA probe
We always use the cDNA probe of approx. 1000 bp in length depending on type of cDNA. Any repete sequences in the probe could make a strong background using TSA.
- prepare at least 2 μg of amplificate of cDNA from reverse transcription reaction by PCR and specific primers
- purify the amplificate through agarose gel and column of Gel extraction kit (QIAGEN)
- label this cDNA by Digoxigenine-11 dUTP alkali stable (Roche) and Deca Label Labelling kit (Fermentas) – labelling by random primers. We always label 1μg of cDNA for 20 hours. After this time we purify the probe with column of gel extraction kit (QIAGEN) without using an agarose. Just mix probe with binding buffer. Elution is done with 50 μl of sterile water. We always use 2 μl of probe in 50 μl reaction per one slide.