When the RNA probe was used, the air-dried chromosome squashes were treated with RNase A (0.6 mg/ml) diluted in 2X SSC at 37 °C for at least 2 h in a moist chamber. The probe mixtures consisted of 50% formamide, 2X SSPE, 0.1% SDS, 30 ng/µl labelled DNA or 200 ng/µl insect RNA mixed with poly (r)-U. Six µl of probe mixture was applied to each air-dried slide and covered with a plastic coverslip. Metaphase and polytene chromosome slides were steam heated at 75 °C for 10 min to denature both chromosomal DNA and probe, and then kept in a closed box at 37 °C overnight (up to 48 h for metaphase chromosomes) for hybridisation. When DNA probes were used, chromosome squashes were treated with RNase A (0.6 mg/ml) diluted in 2X SSC at 37 °C for at least 2 h in a moist chamber. Hybridization procedures and detection of DNA labelled probes were described elsewhere1720. When the RNA probe was used, the slides were washed twice in 0.1X SSPE, 0.1% SDS at 37 °C, followed by incubation at room temperature in 1X TBS, 0.1% Triton X-100 (TBST), 10% Superblock (Pierce) for 30 min. The slides were incubated with goat IgG anti-RNA.DNA hybrid22 diluted 1:50 in TBST/Superblock solution. After 1 h at room temperature in a moistened chamber, the slides were washed twice in TBST for 30 min and then incubated with rabbit IgG anti-goat labelled with TRITC (Sigma) diluted 1:200 as described above. The slides were washed twice in TBST for 30 min and finally once in 1X TBS for 5 min. Chromosomes were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The slides were mounted in antifading medium (Vectashield, Vector Labs) and inspected with epifluorescence optics (Nikon).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.