Keeping check of an In-situ hybridisation experiment in general and making good
Posted 17 September 2009 - 02:51 AM
First some random (or not so random) thoughts:
From my undergraduate degree in biochemistry I learned that to check yield/efficiency etc. of each step in a long protocol (i.e. purification) is golden.
Now In my graduate research Im facing whole-mount In-situ hybridisation. Itīs a long protocol and it seems rather "black-boxy" to me. See, you only get to kno wheter the experment worked or not after a weeks worth of work. And then you have all the parameters to play with at once without knowing which "failed"! No tweaking of individual incubation times/temperatures or such with immediate feedback as in biochemistry.
As such I will know refere WMISH as the "black-box" experiement
So to the work at hand, with this background in consideration:
I want to make good hapten ssRNA probes (about 1kb, from PCR products of plasmids), but how do I know the probes are good?
I dont want to have to go through several weeks of ISH with probes that dont function to begin with!!!
Posted 17 September 2009 - 04:19 PM
Posted 22 September 2009 - 04:15 AM
After checking around the department dot/blot seems to be the standard way to do it...
Nevertheless, what cought my attention was an approach that involves doing a regular EtBr agarose gel with the 1. starting DNA, 2. DNA+Synthesis (adding a new RNA band), and 3. DNA+Synthesis+DNAse (removing the DNA band).
The gel should be run at high voltage, reducing time for rnases to work, and with as clean as possible reagents.
The problem i see from this are the famous RNAse-ses, but then again everyone seems to have diffrent opinions on thoose..
Posted 23 September 2009 - 02:21 PM
Posted 06 October 2009 - 01:21 AM
That would make my ISH flourescent ISH right?
..or do you mean to combine both the hapten (Ab-binder) and the FITC?ŋ
PS. And that woul'd be seen by the naked eye?ŋ