373A AND QIAPREPS

[Martin D. Leach]

In article <3ht6h5$pdk at mserv1.dl.ac.uk>, <I.Ridgers at nhm.ac.uk> wrote:

> HI,
> 
> COULD SOMEONE TELL ME IF IT IS NECESSARY TO RESUSPEND DNA IN WATER FOR THE
> PURPOSE OF T7 (SEQUENASE) AND CYCLE SEQUENCING AS SUGGESTED AFTER THE
> ALKALINE LYSIS/PEG PRECIPITATION, OR CAN SAMPLES BE RESUSPENDED IN TE
> WITHOUT CAUSING ANY ADVERSE EFFECTS TO THE SEQUENCING REACTIONS.

TE or water have worked well for me in the past
 
> THE PROBLEM IS THE ELUTED SAMPLE DID NOT FREEZE AT -20 C, SUGGESTING
> THERE'S POSSIBLE CONTAMINATION (PROBABLY ETHANOL) FROM THE FINAL WASH
> BUFFER.

hmm.. elute sample in water,,,try evaporating away the final eluted sample
(to get rid of alcohol)...dry it down then resuspend in water.... 
when making the denatured dna sequencing pellet...perform 2x 70% ethanol
washes to get rid of any extra salt..

> HAS ANYONE ELSE HAD PROBS. WITH THIS SET-UP ? AND AM I GOING TO HAVE TO
> RESORT TO ISOPROPANOL PPT. INSTEAD OF USING THE QIAPREPS.

havent used this exact setup...just giving general tips
> 
> IAN
> 
> Ian Ridgers
> The Natural History Museum
> Cromwell Rd.
> London. SW7 5BD

Are you trying to sequence dinosaur DNA??..

Martin

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