Saturday, February 26, 2005

Chimaeric clones


Dr. Fairbanks has pretty much finished with his analysis of my sequences.

Here is the verdict:

14/20 have multiple inserts
That is a lot. That is way more than even I in my worst imaginings thought could go wrong. That means that probably none of my primers are amplifying what we thought they were.

What does that mean for 6 sequences used for phylogenetic analysis? Where they chimaeric?
If yes, then the analysis is bunk.

If no, Then maybe, assuming there isn't any other HUGE messups then the analysis is legitimate.

What does that mean for the minisatellite sequences?

There were thirty something of those. We need to check the area used in the alignment, only 80 bases. Pretty good chance those 80 bases are intact.

Solution: Submit the consensus sequence to Genbank.

What does that mean for the FISH data (Microscopy pictures)?

IF the clones used are not chimaeric then it too is OK, if not then it also needs to be redone.

What does this mean for the microsatellites?

14/20 are probably not reliably amplifying from one region.

That is a huge problem, especially considering the number of short repeat sequences.
1. So the key with those might be to pick the best of the repeats and go over the sequences to see if they have multiple inserts.
2. Keep the ones that are good and
3. test to see if sequence size is consistent in 'Real'.
4. Test for polymorphism in other entries in panel used by Shanna.

Ultimate solution.

Two words. Mental breakdown

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