Minipreps for ABI sequencer

Robert Hindges hindges at vetbio.unizh.ch
Tue Oct 4 18:48:35 EST 1994


In article <1994Oct3.170011.10298 at news.unige.ch>, maria at divsun.unige.ch
(???? maria) wrote:

>   Does anybody have a good and cheap protocol for plasmid minipreps 
> for sequencing on the ABI for dye primer sequencing?
>  Thanks in advance
> 
> *******************************************************************
> Maria Lalioti            
> Division of Medical Genetics       tel (Switzerland) (22) 702.56.99
> CMU, University of Geneva          fax (Switzerland) (22) 702.57.06
> Geneva, Switzerland                email maria at medsun.unige.ch      


I used the following protocol, which worked fine:

1. Pellet 1.5ml culture for 1 min in microfuge
2. Remove s/n and resuspend the pellet in 200ul GTE buffer by pipetting up
an down
3. Add 300ul freshly prepared 0.2N NaOH/1% SDS and mix the contents of the
tube by inversion and incubate on ice 5 min (do not vortex throughout the
whole procedure; do not incubate with NaOH longer than 5min)
4. Neutralize the solution by adding 300ul of 4M NH4-acetate mix the
contents of the tube by inversion and incubate on ice 5 min.
5. Remove cellular debris by centrifuging for 10 min at roomtemperature!
and then transfer the s/n to a clean tube
6. Add RNase A (DNase free) to final concentration of 20ug/ml and incubate
at 37¡C for 20 min.
7. Extract once with 400ul phenol/chloroform 1:1, once with 400ul
chloroform.
8. Precipitate the DNA by adding 600ul of 100% isopropanol and 100ul of
7.5M NH4-acetate and immediately centrifuging for 10 min at
roomtemperature.
9. Wash the DNA twice with 500ul of 70% ethanol and then dry under vacuum
for 3min.
10. Dissolve the pellet in 10ul of water! and use 2ul for one sequencing
reaction. You can check 1ul on a agarose gel.


If you do other miniprep protocols, follow these instructions:
- precipitat DNA only at roomtemperature
- do not incubate with NaOH longer than 5min
- make RNase digestion and phenol/chloroform extraction
- be shure to remove phenol completely
- be shure to remove any salt completely, dissolve DNA in water
- wash DNA with 70% ethanol
- after the preparation:precipitate the DNA with same volume of NH4-acetate
and 3 volumes of isopropanol to remove fragments shorter than 100bp.


We also used the QIAwell from Quiagen following their standard protocol. It
also worked fine.


Robert Hindges             



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