help TA cloning!

Wolfgang Schechinger hubahopp at gmx.de
Fri Dec 20 04:09:10 EST 2002


Emir, 

On paper your assumption is right. In reality, I found that background
becomes a little lower. There are always some plasmid individuals that
won't follow the rules: They just get one cut. And dephosphorylation lowers
the chance of them religating. If you bought SAP once an now have 1 ml
sitting in aliquots in the freezer, you probably have so much of it that
you need to worry more about the cost of the pipet tip you will be using
for dispensing :) 

If you want to decrease background further but need to perform a shaking or
mixing procedure afterwards, transfer the digest without disturbing the
liquid to a new cup and do the workup there (e.g. phenol extraction). It
seems that some DNA gets stuck in corners while mixing and becomes
inaccessible to enzymes. As soon as you shake the container, Murphy's laws
will apply. 

Normally, all that might not be necessary, but when you need extremely low
background...

Wolfgang



At 04:57 PM 2002/12/19 -0600, EK wrote:
>
>"Wolfgang Schechinger" <hubahopp at gmx.de> wrote in message
>news:3.0.6.32.20021218103436.0081fca0 at pop.gmx.net...
>
>...
>> - digest vector with the same 2 favourite enzymes and include some SAP(aso
>> in one RxN)
>> - spin column purify vector and insert, EtOH prec.
>> - ligate, transform, check.
>>
>> Usually works like a charm, except on mondays past full moon.
>>
>> Wo
>>
>Would adding SAP really make any difference? I always thought
>dephosphorylation is not necessary if you digest with 2 enzymes that produce
>non-compatible overhangs? SAPing would certainly be necessary in case of 2
>blunt cutters though.
>Emir
>
>
>

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