PCR Ligation Issues!!!

ChenHA hzhen at freeuk.com
Thu Sep 29 07:06:52 EST 2005

On Wed, 28 Sep 2005 22:15:08 GMT, dk at no.email.thankstospam.net (DK)

> In case you have not noticed, a lot of cloning these days is done 
>incompetently by people who are semi-competent to do it to begin 
> with. 

Yep.  I can tell you about the people who claimed to have done cloning
but couldn't not even do simple molecular biology procedures properly
like doing DNA prep (using kits!) or making competent cells and do a
simple transformation.  

> When something becomes a common place, it simply has to 
> be done and ASAP, and the quality and real training becomes less
> important . Classic examples are hideous looking gels and westerns
> that now became a norm in even most prestigious journals and the 
> fact that in many places grad students cannot pour a decent gel 
> simply because they never had to. Same with cloning. 

I think a lot of the skills are slowly disappearing.  That's partly
progress I guess, for example I used to be able to do the most
beautiful DNA sequencing gel, but how many people do their own
sequencing nowadays?  All I do now is just moan about how awful the
sequencing results I sent off to be done are.

>         The TOPO kit really did boost productivity on our lab a lot. 
>That's just the fact. I normally direct double digest my products 
>but always leave out 2 ul of PCR reaction to do TOPO in those
>*rare* cases when the normal cloning did not work. With those, I 
>don't bother to screen colonies - when there are "enough" colonies,
>it's always > 95% correct insert. 
>        As for the price, yes it is ridiculously expensive. We use 1/2 of
>everything so that one kit lasts 50 reactions instead of 25. 

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