Difficult to clone

Danuta Bois dbois at netsrq.com
Mon Oct 9 13:04:30 EST 1995


gxingnia at extri.ucc.su.oz.au (Xingnian  Gu) wrote:

>Hi netter,

>Our lab is trying to clone a 3kb virus DNA to pGEM plasmid.  But most 
>of the time there was no recombinant clone could be found, enen we 
>could get many white colonies. Could we borrow some body brian to find 
>out what is going on ?

>The protocol we followed is Promega' manual.

>Thanks in advance !


>David Z.

Can you give us more information about the exact protocol you used,
and the steps involved?  Also, what is the nature of the viral DNA
you used for cloning:  was it purified linear viral DNA or a mix of
restricted genomic plus viral DNA?  

Did you get a lot of white colonies, or just a few?  If you used a
genomic plus viral DNA mix, you may need to screen hundreds of
colonies before you find a viral clone.

Did you check some of those white colonies to see if any had inserts
and if so, how long?

It's hard to guess what happened without knowing exactly what you did.

Cheers,

Danuta Bois




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