This is a repeat since the previous attempt at obtaining sufficient quantities of plasmid for sequencing failed. Although I’m not sure why, I figure it’s easy enough to re-do using ampicillin stocks that aren’t many years old.
The old ampicillin may not have been strong enough to put enough selective pressure on transformants, which possibly led to such little plasmid recovery.
I prepared fresh ampicillin solution (20mg/mL) and made new LB plates (ampicillin concentration 100ug/mL).
Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).
Cells were plated on pre-warmed (37C) LB Amp100 plates.
Plates were incubated overnight at 37C.
No transformants. So, this suggests that the original ampicillin was bad. Now, the lack of transformants suggests the plasmid concentration is too low. Will try eluting the DNA from the second spot of Whatman paper.