Fig 8 (FISH guide)
LegendFig. 8.Hybridization efficiency and background control.Three different cosmids, each detected with a different color were hybridized on a set of slides, after mixing 1 µl each probe with 200 ng human Cot1 DNA. These slides were aged using dry heat, 8 hours at 65 C, then pretreated 10 minutes in pepsin (some slides), denatured 2 minutes and hybridized overnight. The low amount of competitor DNA allowed repetitive sequences to bind and increased background. Slides were detected using the scheme from Table 7. a)andf) show the reference hybridization, which took place overnight at room temperature, and was done on a pepsin-pretreated slide. 1 µg of Cot-1 DNA was used in the hybridization. b) 3% BSA was used to block unspecific binding sites on the slide. The background level did not significantly decrease compared to the reference. c) Hybridization was done overnight at 37 C. Results were not significantly better than the reference. d, e) Non-pepsin pretreated slides show a decrease in signal size and intensity compared to the reference, indicating that protease pretreatment improves hybridization results. g) A longer denaturing time (6 minutes) did not improve FISH results. h) A shorter hybridization time (3 hours) at room temperature produced results similar to the reference, indicating that 10-20 ng of a cosmid probe do not need to be hybridized overnight. i) A temperature-variable hybridization was performed by placing the slide on a metal block of a thermocycler and running 15 cycles of 10 minutes at 60 C and 10 minutes at 15 C each. Signal to noise ratio was best of all slides. j) Hybridization in the presence of 1 ?g Cot1 DNA. k) Hybridization in the presence of 10 ?g salmon sperm DNA. Last two slides show that Cot1 is a much better competitor of repetitive sequences than salmon sperm DNA. l) Hybridization in the presence of 1 ?g Cot1 DNA and 10 ug salmon sperm. Results are better, indicating that the more overall amount of competitor DNA, the better the signal to noise ratio of the hybridization.
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Last modified on: Feb12, 2001