This was a PCR assay using two primers sets mixed together into one mastermix.  Specifically, one primer set for the efflux RND transporter subunit and the other for Lanthionine synthase C family protein. We use these primer sets as a cost-effective way to screen S. aureus strains before sending them in for sequencing.

 

PCR was run normally on the thermocycler. (Attached the protocol)

 

Sample breakdown:  These are different clonal variants of staphylococcus aureus. The band showing up at 501bp is from a sequence type 5 (ST5) sample while the band showing up at 308bp is a ST8 sample. Blanks (negative controls) were also run with only mastermix (No bacterial DNA).

 

4ul of gel dye was mixed into each sample (Tip was changed here between samples).

 

I then aliquoted 40 ul of DNA 1kb Trackit Ladder onto a small piece of parafilm. (This was so I didn’t have to keep going back into the ladder tube)

 

15ul of each sample was added into the wells left to right with ladder dividing up each replicate. Between samples the pipette tip was rinsed 3-5 times in the running buffer at the bottom of the gel running apparatus. 

 

The gel was then run for about 60 min at ~130V

 

Protocol: Full Colony PCR Protocol

The sample used was DNA extracted from a mouse tail snip. The DNA used for all 5 replicates came from the same extraction preparation. The PCR product for this particular reaction should be ~190 bp.

PCR was performed following the attached protocol and general guidelines from New England Biolabs using their standard 10X Taq Reaction Buffer for 25ul reaction volumes. After PCR, DNA loading dye was added to each replicate of sample. A blank sample was created using only DNA hydration buffer (10mM Tris, 1mM EDTA) and loading dye.

Ladder used was Quick-Load 100bp ladder from New England Biolabs. Samples were loaded onto a 1.5% agarose gel as follows, with the same tip being used for each wash group:

 

4ul of ladder – WASH – 15ul Sample – WASH – 15ul Blank

This procedure was performed using either 1, 2, 3, 5, or 10 washes.

 

Gel was run for 65 minutes at 120V, and stained with Ethidium Bromide solution from BioRad before being imaged in UV gel transilluminator.

Absence of ladder in the sample lane, and absence of product band in the blank lane provides confidence that washing the same tip in-between samples during gel loading may be a useful way to save tip waste.

 

Protocol: Full NEB 10x Taq Polymerase Protocol