help needed with cosmid prep!!!

Andre Hamel hamel at ccu.umanitoba.ca
Wed Apr 7 11:32:17 EST 1993


How long are you PEG ppt'ing? ... on ice too? And how fast/long are the
PEG ppt'ns being centrifuged?

At least a couple hours on ice (overnight's good too), spun at 8-10,000
rpm for at least 10 min (30 min. okay) ... "pellet" might not be very
"tight" or firmly adhered to tube, thus caution needed. ALSO, ARE THE
BUGS GROWN OVERNIGHT UP TO "HIGH enough" density ... pseudoamplified,
also what is origin of replication of your cosmid vector ... pBR322/pUC
type replicon ... perhaps chloramphenicol or spectinomycin treatment of
cells (see Maniatis) for cosmid amplification with some of the supernatant


regards

Andre Hamel
Manitoba Vet.Virol.
Winnipeg, Manitoba
Canada
email: hamel at ccu.umanitoba.ca

In article <1993Apr7.150051.7540 at gserv1.dl.ac.uk> ahill at crc.ac.uk (Mr. A.F. Hill) writes:
>
>hi
>
>ive been trying to grow up a cosmid (~45kb) and have had varying results
>using different methods. the problem is that if i get DNA i cant cut it with
>restriction enzymes. i have used the standard alkaline lysis / PEG precipitn
>methods and also the triton lysis method. i can cut the dna from peg pptn
>but from a 500ml prep i only get 1-5 ug DNA which is a pretty low yield.
>
>ive noted that most of the dna is lost at the peg/nacl pptn stage as when
>i reprecipitate the supernatant there is heaps of dna left - any reason
>why this is happening?
>
>hope this makes sense to someone!
>
>any suggestions welcomed,
>
>thanks
>
>Andy
>
>==============================================================================
> Andy Hill				  |		
> Prion Disease Group			  |     E-Mail: ahill at uk.ac.crc	
> Dept. Biochemistry & Molecular Genetics  |-----------------------------------  
> St Mary's Hospital Medical School	  |     Phone: 071 723 1252 ext. 5469
> LONDON W2 1PG				  |     Fax:   071 706 3272
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