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(apparently) Foolproof method for verifying a yeast locus by PCREdit

Used, for example, to verify that a deletion (and insertion of a selectable marker) has been achieved. The following strategy for verification is used: Dna

where RHO2 was deleted and replaced by kanMX4 (the standard selectable marker for the original yeast deletion collection). The black region is the 5'-flanking region of RHO2 and its 3'-flanking region is blue.


Two separate PCR reactions are done on a standard Smash n' Grab DNA prep of the strain: one using the primers rho2ver-1 and KAN-K and the other using KAN-C and rho2ver-2.


rho2ver-1 and rho2ver-2 were designed so that their melting temperature matches those of the two standard KAN primers (which is 52 deg, according to MATE, the software used).


Here's what I did for RHO2:


1) Made the GCK file shown above (called rho2;;kanMX4) by deletion the RHO2 ORF from the file "RHO2 1+1" (i.e. containing 1 kb on either side of the ORF, from SGD) and pasting in the sequence from GCK file kanMX4int.


2) Set the KAN-K primers right end to zero so can easily see when you are ~1000 bp leftward of it. Do a similar thing on the right to pick the ver-2 primer.


3) Pick a likely-looking stretch >16 bp long that may be your primer. Paste it into the 'M'erged 'A'nnealing 'T'emperature 'E'stimation software (at http://protein.bio.puc.cl/cardex/servers/melting/) using the default setting and if it's within 2° of 52° proceed to next step...


4) ...which determines if there's excessive secondary structure. This is at MFOLD (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi). If more than 3 contiguous base pairs are capable of forming within the primer, start over. If not, you're done with primer design.


5) PCR reaction contains

one primer (0.1 mM)

1 ml

other primer (0.1 mM)

1 ml

Biolase Taq

1 ml

10x Biolase buffer

10 ml

50 mM MgCl2

3 ml

10 mM (each) of dNTP soln.

3 ml

SnG

1.5 ml

water

79.5 ml

and each reaction is 30 ml (that's as low as I've ever gone, but perhaps it can be smaller).


I originally wrote this on 12/28/09, and am leaving it as is, although I'd bet it works using Mark's colony PCR protocol (easy) as well as with Smash n' grabs (hard).


--Dan Lockshon 8/12/10