User:Jarle Pahr/Verification

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Notes on how to perform experiment verification and controls, including ideas/procedures for verifying successful cloning:

To facilitate colony PCR screening, add a "barcode" sequence to the insert, upstream of the target sequence. Colony PCR can then be performed with a reverse primer binding to a region in the vector downstream from the insert site (to avoid having to add a barcode sequence downstream of the target sequence), and a forward primer binding to the upstream barcode site.

As an alternative, use two primers binding to the backbone: One before the insert and one after the insert. In this way, the size of the region between the primers (which should contain the insert) can be estimated after PCR.


Options for verification by restriction digestion:


Digest with an enzyme with restriction site only in the insert (Will cut only if the cloning was successful).

Digest with an enzyme with restriction site only in the (Will discern between original, uncut plasmid and any derivates)

Digest with an enzyme with restriction sites in both insert and backbone (will give different number of fragments)


Implicit controls:

Consider possible situations where an explicit negative control is not needed. Can failure-clustering be used to discern the origin of failure? Example: Parallel cloning reactions using different inserts but same backbone. For a large number of reactions, can statistics be used to "prove" that the backbone by itself does not yield transformants?

Temporary conclusion: An explicit negative control is much less work!


Types of verification screens:


Direct sequencing: Confirm the presence of correctness of a sequence.

Most relevant for: Plasmids.

PCR: Confirm that a short sequence, or pair of sequences, is present in some genetic material.

Most relevant for: Strains

Phenotype verification: Check that the expected phenotype is displayed.

Most relevant for: Strains and strain/plasmid combinations.

Software:

Genoread sequence verification: http://www.genoread.org/

http://www.cloneqc.org/

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