373A AND QIAPREPS

I.Ridgers at nhm.ac.uk I.Ridgers at nhm.ac.uk
Wed Feb 15 10:27:33 EST 1995


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.

THE MAIN REASON FOR THIS QUESTION IS THAT I HAVE JUST STARTED USING THE
QIAWELL PLUS MANIFOLD SYSTEM, WITH THE QIAPREP STRIPS.

THE QIAWELL PROTOCOL SUGGESTS FINAL ELEUTION USING TRIS/HCL, THE CATALOGUE
AND THE REP SAY THAT ELUTION CAN BE DONE IN WATER.

THE PROBLEM IS THE ELUTED SAMPLE DID NOT FREEZE AT -20 C, SUGGESTING
THERE'S POSSIBLE CONTAMINATION (PROBABLY ETHANOL) FROM THE FINAL WASH
BUFFER.

SUBSEQUENT TAQ AND T7 REACTIONS WERE ONLY 25% SUCCESSFUL FROM THESE
'PURIFIED' SAMPLES.


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.

THANKS

IAN

Ian Ridgers
The Natural History Museum
Cromwell Rd.
London. SW7 5BD

E-mail ir at nhm.ac.uk
Tel. 071 938 9297

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