Daniel:Notebook/HiResChrPaint/2013-12-13: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=FISH Parameters= From some of my background research on FISH protocols, it seems there is a lot of parameter space for me to explore with these protocols. So far, I haven't...") |
>Djacobse No edit summary |
||
Line 5: | Line 5: | ||
*(1) '''Stringency of hybridization/wash'''. Various protocols call for various levels of formamide, temperature, and buffer concentrations. The stringency of these protocols is defined by formamide concentration (higher concentration, more stringent), by temperature (higher temperature, more stringent), and salt concentration in the buffer (lower salt, more stringent). By looking around at various protocols, I'd say I'm currently using a stringent protocol. Stringent hybridizations yield lower binding percentages (due to increased need for sequence specificity) and more stringent washes wash out increasingly high levels of probe specificity (with really stringent washes only perfect matches remain). From the general results of the [[Daniel:Notebook/GenomeMiner|HL155 sequencing project]], it would appear that there are 1-2 errors per probe, which would indicate I need generally less stringent conditions. | *(1) '''Stringency of hybridization/wash'''. Various protocols call for various levels of formamide, temperature, and buffer concentrations. The stringency of these protocols is defined by formamide concentration (higher concentration, more stringent), by temperature (higher temperature, more stringent), and salt concentration in the buffer (lower salt, more stringent). By looking around at various protocols, I'd say I'm currently using a stringent protocol. Stringent hybridizations yield lower binding percentages (due to increased need for sequence specificity) and more stringent washes wash out increasingly high levels of probe specificity (with really stringent washes only perfect matches remain). From the general results of the [[Daniel:Notebook/GenomeMiner|HL155 sequencing project]], it would appear that there are 1-2 errors per probe, which would indicate I need generally less stringent conditions. | ||
*(2) | *(2) '''Failure to penetrate cell membrane'''. There is a chance that the probes are simply not getting into the cell, or in to the nucleus. To test the nucleus, I could use a protocol to completely eradicate the cell membrane (like a lysis buffer used in MIDAS or something. However, first I can test using a 70% ethanol rinse followed by a 0.1M HCl mix, which is what Hosuk and Matt use for RNA FISH. |
Revision as of 23:06, 13 December 2013
FISH Parameters
From some of my background research on FISH protocols, it seems there is a lot of parameter space for me to explore with these protocols. So far, I haven't had any luck on my FISH experiments, and I think one of two factors are the most likely responsible:
- (1) Stringency of hybridization/wash. Various protocols call for various levels of formamide, temperature, and buffer concentrations. The stringency of these protocols is defined by formamide concentration (higher concentration, more stringent), by temperature (higher temperature, more stringent), and salt concentration in the buffer (lower salt, more stringent). By looking around at various protocols, I'd say I'm currently using a stringent protocol. Stringent hybridizations yield lower binding percentages (due to increased need for sequence specificity) and more stringent washes wash out increasingly high levels of probe specificity (with really stringent washes only perfect matches remain). From the general results of the HL155 sequencing project, it would appear that there are 1-2 errors per probe, which would indicate I need generally less stringent conditions.
- (2) Failure to penetrate cell membrane. There is a chance that the probes are simply not getting into the cell, or in to the nucleus. To test the nucleus, I could use a protocol to completely eradicate the cell membrane (like a lysis buffer used in MIDAS or something. However, first I can test using a 70% ethanol rinse followed by a 0.1M HCl mix, which is what Hosuk and Matt use for RNA FISH.