Sequencing from Minipreps - Is it possible to sequence directly from minipreps? (Nov/28/2006 )
Alkaline lysis minipreps should not be used directly for sequencing because they can contain contaminants that inhibit the sequencing reaction or degrade the capillaries in the sequencer.
The problem i got is, from miniprep, the concentration is low and thus not sure the purity by measuring A260 and 280. Have tried once to sequence with miniprep sample, bad result. so i always use either midiprep or maxiprep products for sequencing. Results are always good but then i need to spend more time...
Any suggestion?
If you are getting so little DNA from your miniprep, then you are doing something wrong with your procedure. Try to redo it, you should have no trouble getting a couple of ug of dna from this.
Not necessarily. If the plasmid is a low copy number plasmid or the host strain does not produce large yields of DNA the concentration may be lower then expected. This can be countered by growing several cultures of the same clone at once and pooling the sample to increase concentration. Having said that, minipreps usually produce enough DNA for good sequencing
next time for sequencing using miniprep sample i will follow "perneseblue"......if the result is good then its a great relief as i will save atleast 1 hour which i spend for peg treatment
miniprep by alkaline lysis method for sequencing is not the total same as for usual DNA mini prep. so no need to be worried about the quality of the DNA , it will be fine if you treat properly. usually i got a lot of DNA by miniprep to do sequencing.........then i check the conc by running in gel and compare the band intensity with a standard (obviosuly it is a "about/approximate" measurement)
if u failed to do sequencing then u must have some problem when u prepare DNA by miniprep