Sequencing from Minipreps - Is it possible to sequence directly from minipreps? (Nov/28/2006 )

Hi,

I have some cloning minipreps, which I have been sequencing by doing an m13 pcr followed by the normal cycle sequencing reaction.
However, is it possible to sequence directly from these minipreps?

Depends on the purity of the preps. You might get funky results if they are not clean enough.

usually DNA sequencing from alkaline lysis minipreps is not good.
I've obtained good results only by columns miniprep.
If you can do a midiprep by alkaline lysis method, this one will be opk for sequencing. I suppose it's because of RNAse/proteinase K and PEG/NaCl treatments...

I used a Qiagen miniprep kit. DO you have to do anything to the minipreps before doing the cycle sequencing reaction (apart from maybe cleaning or something)?

Sequencing after normal protocol from Qiagen miniprep is OK, done it often without any specials, check concentration of DNA of course.

There's an extra step in the sequencing reaction, to melt the plasmid.

Patty

p.s. I tried doing just what you're talking about with poor luck. We didn't troubleshoot. We went back to M13 sequencing off the M13-amplified product. We have also found that sequncing with an internal primer, off the PCR product, worked well. PCR product from miniprep DNA can be more robust than PCR product from cells.

i've had sequencing work with both qiagen column miniprep and alkaline lysis method. Just make sure you're DNA is clean. I have never had to melt the plasmid before hand, always got good results. I guess it depends on how robust the sequencing protocol is.

V

I agree with Vetticus. I have sequenced from qiagen column miniprep many times. You just need to check the DNA concentration so you can add the appropriate amount to the sequencing reaction. Apart from that..just follow the sequencing units sequencing protocol. Also, never had to add an extra melting step.

i have done sequencing usign the DNA sample made by alkaline lysis mehtod. but for prepare the sample for sewuencing we treated the sample with peg

and to add to the story

I have done sequencing on 2ml miniprep alkaline lysis. Avoided PEG. Unless you throughly remove PEG with a good 70% EtOH wash, PEG will cause the sequence read to be poor. (I never did manage that trick well, so PEG was avoided)

Did 2 rounds of phenol/Chloroform followed by ethanol percipitation and a 70% ethanol wash.

Sequenced... had enough DNA for about 2 reads.