Daniel:Notebook/HiResChrPaint/2013-8-5: Difference between revisions
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[[Image:Fish-hybridization-temp-compare.png|600px|thumb]] | [[Image:Fish-hybridization-temp-compare.png|600px|thumb]] | ||
For a good image, the signal should be distinctly above background, but there will likely be background present in the nucleus. Second, also in the thumbnail, | For a good image, the signal should be distinctly above background, but there will likely be background present in the nucleus. Second, also in the thumbnail, it looks like 42 C incubation will be better than 37C. Note that both images are from 16 hour incubation and human stained cells, just as I use. I also found more points, and I will summarize them all here: | ||
*Incubate at 42C instead of 37C; use water bath for this | |||
*Denature using the slide apparatus of the thermocycler we have in lab; can get to 92C easily and more even denaturing than water bath | |||
*Use human cot-1 DNA (would have done this for the last ones but we didn't have any | |||
*Use fewer probes (10-20 pmole total). The [[Daniel:Notebook/HiResChrPaint/2013-8-1|previous FISH experiment]] used 20 pmol of each probe. Will hopefully reduce background to use less probe | |||
*Use a negative control; maybe hybridize with just the human cot-1 DNA (plus everything else in the hybridization cocktail except probe) | |||
==Lambda Exonuclease Digestion== | ==Lambda Exonuclease Digestion== |
Revision as of 19:15, 5 August 2013
S1 Probe Preparation (Started 8/2/2013
Researched this morning on new ways to improve the FISH protocol. First of all, I noticed that in the supplementary material of Beliveau et al the images are not so crystal clear as the paper (go figure). From the supplements, I see that nucleii seem to have more background than I originally suspected (see thumbnail).
For a good image, the signal should be distinctly above background, but there will likely be background present in the nucleus. Second, also in the thumbnail, it looks like 42 C incubation will be better than 37C. Note that both images are from 16 hour incubation and human stained cells, just as I use. I also found more points, and I will summarize them all here:
- Incubate at 42C instead of 37C; use water bath for this
- Denature using the slide apparatus of the thermocycler we have in lab; can get to 92C easily and more even denaturing than water bath
- Use human cot-1 DNA (would have done this for the last ones but we didn't have any
- Use fewer probes (10-20 pmole total). The previous FISH experiment used 20 pmol of each probe. Will hopefully reduce background to use less probe
- Use a negative control; maybe hybridize with just the human cot-1 DNA (plus everything else in the hybridization cocktail except probe)