plasmid extraction...

[fibonacci]

when i do the plasmid extraction by using published method by Oloni Kotchoni et al. (2003), i didn'y get any band when analyzed by 1% agarose gel...but when i measure by spectrophotometer, the concentration of the 'plasmid' is quite high, ~1000ng/ul and the purity is quite good...

 

but then, i do PCR colony juz to check the insert of my cDNA..but, i still didn't get any band in the gel picture...

 

actually, this is the 2nd time i do the electhroporation..the 1st time i do it by using same method, i was successfully obtain the cDNA...but the 2nd time, i fail to observe the cDNA in my plasmid...

 

so, any suggestion @ advice for me....

 

p/s - is there any effect after i do the electroporation, i incubate the cell at 37deg C for three hours????the protocol recommended to incubate at least 90 minutes...

[CharonY]

I cannot find the paper in question, however the given concentration should be easily visible on the gel. Something is definitely off. Even if you only measured chromosomal DNA it should be somewhat visible on the gel, unless it is totally sheared and ran out of it.

Maybe you could post the photometre readings? Did the PCR on the insert worked before? If so it is pretty much proof that you got not plasmids.

 

Regarding the incubation at 37°. This incubation step has two functions. First, it allows the cells to regenerate, but more importantly, it also allows the cells to express the resistance genes that are on most vectors. However, around 45 minutes (around one generation time) usually suffices. Some incubate longer to artificially increase the transformation rate (as cells are multiplying), but I generally would not recommend to do so. Incubation for three hours, is too long though. As it is done under non-selective conditions the cells might throw out the plasmids again. While they are multiplying.

[protist]

is your antibiotic OK? dumb question I know but to make sure you have plasmids at all