Re: [ccp4bb] regarding cloning

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: regarding cloning
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2008-09-01

Next message:
Subject: snippet of code for coordinate fit
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-09-02

Subject: Re: regarding cloning
From: Brian Wengerter bwengert {- at -} AECOM {- dot -} YU {- dot -} EDU
Date: 2008-09-02

Alternatively, you could skip troubleshooting digestion/ligation/etc.
and use a kit based on site-specific recombination, like the In-Fusion
kit that Clontech sells. (I don't have any financial interest here--I'm
just a graduate student, but I've had good results using it.) The kit
is not without its own downsides--it's a bit pricier than traditional
cloning, you'd have to design another set of specific primers, and you
have to be very careful about the vector:insert ratios you use.

Good luck,
Brian

Artem Evdokimov wrote:
>
> Hi,
>
>
>
> First of all -- I am curious why did you decide to put in an extra
> step (the T/A cloning into an intermediate vector)? You can happily
> digest your PCR product with NheI/BamHI, clean up and ligate into the
> appropriately digested pET-23a(+). If you have issues, you should
> definitely try this.
>
>
>
> Now, since you do have an intermediate step -- did you verify that
> everything was OK after havig subcloned your insert into whatever
> vector you're using? Did you sequence the insert and most importantly
> did the sequencing confirm the nature of the linker regions?
>
>
>
> The enzyme pair that you chose has a slight issue with digestion
> buffer -- most people would choose NEB buffer 2 (since buffer 3 is bad
> for Nhe) where Bam still has '100% activity' -- however, in buffer 2
> you can have star activity of the Bam due to the somewhat lower salt
> concentration (50 mM instead of the optimum 100 mM). It's not
> impossible to imagine that you have issues with digestion. This can be
> easily avoided by sequential digestion although of course it's
> slightly more work (but if you cut out the T/A cloning step that's
> actually still faster).
>
>
>
> So, in conclusion the most likely issue is digesiton (probably of the
> pET vector, to be more specific). Next likely issue could be ligation
> -- make sure that you base your ligation ratio on the gel intensity of
> the bands as well as on the OD260 of your DNA. Faulty primers are not
> likely to be an issue since you seem to be able to restrict your
> insert out of the intermediate vector.
>
>
>
> Please note that you can often use SpeI or XbaI instead of Nhe since
> they have compatible sticky ends. Clearly this depends on the vector
> you're working with and I am too lazy to look up pET23 polylinker.
>
>
>
> Artem
>
> ------------------------------------------------------------------------
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
> Of *vijay srivastava
> *Sent:* Monday, September 01, 2008 3:06 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] regarding cloning
>
>
>
> Hi,
>
> I am trying to clone a 1.2kb insert into a expression vector pET 23a
> through T/A cloning. The restriction enzyme used is Nhe1(NEB) and
> BamH1 (NEB) in the forward and reverse primer recpectively. I was
> succesful in subcloning (T/A vector) and getting my insert at 1.2kb
> after double digestion and also the vector at 3.7kb ,for the ligation
> i am using the ratio of vector to insert is 1:3,1:2,getting the colony
> after the transformation but some how when i used to confirm my clone
> through double digestion i am not getting my insert at the correct
> position.Some time in the gel only the size of the vector was there.
>
>
>
> ------------------------------------------------------------------------
>
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>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: regarding cloning
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2008-09-01

Next message:
Subject: snippet of code for coordinate fit
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-09-02