nested deletions.

Michael Holloway ca566 at cleveland.Freenet.Edu
Mon May 10 20:09:59 EST 1993


In a previous article, sd39+ at andrew.cmu.edu ("Sheela U. Donde") says:

>I have been trying to make nested deletions using Qiagen purified, 9.3kb
>construct(pRS316 based)DNA. Iam using the Pharmacia Nested Deletion kit,
>and following their kit protocol.Ihave used four different restriction
>enzymes which create Exo-3 susceptible ends, either alone or in
>conjunction with Sac1 which makes Exo3 resistant end,to linearise the
>DNA.After heat inactivating the enzymes at 65C for 20 mins.I continue
>with Exonuclease3 digestion.I find very little if any (10-12bases per
>min.),digestion, at either 30C, 37C, or 42C,or at NaCl conc. of either
>0mM, 50mM,or75mM.The control DNA provided in the kit is already digested
>with EcoR1 and Pst1, and works very well under the same conditions ,
>i.e. digests about 200-300 bases per min.I have no explanation for this.
>        COULD ANYONE GIVE ME ANY SUGGESTIONS? I AM DESPARATE!

Is there any chance that you've overloaded the Qiagen column?  This has 
been a common problem before they revised/clarified their protocol.

Mike

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E-mail: mhollowa at ccmail.sunysb.edu (mail to freenet is forwarded)
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