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HomeBrew's Content

There have been 3 items by HomeBrew (Search limited from 07-March 20)

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#161644 Cannot clone 2kb insert into pENTR D TOPO cloning vector

Posted by HomeBrew on 24 October 2013 - 12:21 PM in Molecular Cloning

The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid containing the PCR amplicon.


Isn't this what you want?

#161125 Colony PCR positive and Digestion negative?????

Posted by HomeBrew on 10 October 2013 - 07:41 PM in Molecular Cloning

We have found it necessary to patch colonies arising on a transformation plate to fresh media, allow those to grow overnight, and sample them as template for colony PCR to avoid an undue number of false positives.  Didn't know the observation of inordinately high false colony PCR positives was published anywhere, but in my lab, transferring colonies to fresh plates before using them in colony PCR is standard SOP, learned the hard way over many years of such experiments -- the extra day is well worth it...

#160965 q value in rna seq

Posted by HomeBrew on 07 October 2013 - 04:43 AM in ChIP and Next Generation Sequencing

The q-value is an adjusted p-value, taking in to account the false discovery rate (FDR).  Applying a FDR becomes necessary when we're measuring thousands of variables (e.g. gene expression levels) from a small sample set (e.g. a couple of individuals).  A p-value of 0.05 implies that we are willing to accept that 5% of all tests will be false positives.  An FDR-adjusted p-value (aka a q-value) of 0.05  implies that we are willing to accept that 5% of the tests found to be statistically significant (e.g. by p-value) will be false positives.  Such an adjustment is necessary when we're making multiple tests on the same sample.  See, for example, http://www.totallab..../pq-values.aspx.

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