Disappearing Plasmids--Help!

contente at usuhs.usuhs.mil contente at usuhs.usuhs.mil
Wed Aug 17 17:42:20 EST 1994


wrightc at ctrvax.vanderbilt.edu (anon) writes:
>In article <djt2-1608941806040001 at path22285.path.cwru.edu>,
>djt2 at po.cwru.edu (Dennis Templeton) wrote:
>> 
>> I have never been too careful with the boiling prep when using HB101's.
>> Boil 45 seconds, 2 minutes, whatever. My tech wanted to do some blue-white
>> screening, and so switched to XL1 blues (a JM101 derivative, I think). As
>> I told her, she boiled for 1.5 minutes. AND GOT NO DNA. She then looked up
>> the original protocol, and using the same colonies, boiled the next time
>> for 45 secs resulting in tons of DNA.
>>
>I have a question.   Are you sure that the second time, the experiment was
>done properly i.e. boiled for , say 30, 45,60,75,90 seconds, and detailing
>an increasing poof of the DNA?  Otherwise I would say your tech messed up
>another part of the protocol in the beginning.

Back in 1981 or 82, when we first started using the Holmes & Quigley boiling
mini-prep, a student in the lab did just the experiment you mentioned.  It
showed that the 40 seconds mentioned in the original paper is indeed ideal;
DNA yield with either less or more time is lower.  We did introduce one
modification, though. After the 40 second boil, the tubes are plunged into
an ice+water bath to "stop the cooking" before they are spun.  I believe the
technician mentioned above did not make an earlier error.

Sara Contente
Department of Pathology
USUHS



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