Cloning trick

[Nasser Mahna]

Dear All,
I would to ask if there is anybody mastered in working with
restriction and digestion.
I want to clone a pcr fragment inside a binary vector. To do it
efficiently, I want to use a restriction enzyme after ligating vector
and fragment, which cuts the original uncut vector before
transformation.
what method do you offer?
Best regards,
Nasser.

[Jayakumar, R]

You mean you want to linearise the construct by cutting it with a RE outside the expression cassette before transformation??? Is that what you mean. I don't see the purpose of cutting a vector after ligating vector and fragment to clone a PCR fragment efficiently. It does not make much sense.

Jay

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[David-Paul Minde]

indeed a good trick if compatible with your strategy, in other cases,
simply using high-fidelity variants of enzymes helps to minimize star
activity artefacts by orders of magnitude (according to published
reports for some NEB enzymes)
best wishes/merry x-mas and all the best for the new year,
David

2009/12/23 DK <d...@no.email.thankstospam.net>:

> In article <mailman.16.126151...@net.bio.net>, "Jayakumar, R" <R.Jay...@roswellpark.org> wrote:
>>
>>You mean you want to linearise the construct by cutting it with a RE
>>outside the expression cassette before transformation???  Is that what
>>you mean.  I don't see the purpose of cutting a vector after ligating vector
>>and fragment to clone a PCR fragment efficiently.  It does not make much
>>sense.
>

> It does make sense! Sometimes. In special or very hard cases.
> The original is hard to interpret so I won't comment. But with regard to
> your reply, I can offer two cases where cutting ligation mix makes
> sense:
>
> Say, MCS contains sites ABCDEF and you clone your insert
> by A and E. If, for whatever reason, your ligation worked very
> poorly (but ligase did work), you will mostly get back your original
> plasmid. However, if your insert is not cut with enzymes B and D,
> you could have cut the ligation mix with either B or D (or both).
> Since linear DNA is 1,000-10,000X less transforming, if you get
> any clones after doing this, almost all of them will be positive.
>
> Even if you are doing everything 100% right and you ligations
> work wonderfully, there are several circumstance where doing
> the above makes sense. I had the following cases:
>
> 1. Huge plasmid that needed to be cut with an enzyme that cut
> it incredibly poorly (and also had start activity). Because of the
> size, there was no easy way to separate cut and uncut forms.
> But couple of other sites that would be excised in the positive
> clones cut it without any problems.
>
> 2. Blunt cloning that destroys site for the blunt cutter. (In fact,
> with a little foresight, this can be used to do directional
> blunt cloning).
>
> 3. Quick and dirty protein expression tests when ligations
> work/should work very well. Transform cut ligation mix
> directly into expression strain, don't select clones but scrape
> everything off the plate (which should be >>99.9% positives)
> and use it as a starter culture. This way, the day after ligation
> one can induce and run an expression gel to, say, check
> expression level/solubility pattern of the new construct(s).
>
> DK

>
>
>>-----Original Message-----
>>From: methods...@oat.bio.indiana.edu [mailto:methods...@oat.bio=
>>=2Eindiana.edu] On Behalf Of Nasser Mahna
>>Sent: Tuesday, December 22, 2009 12:23 PM
>>To: met...@magpie.bio.indiana.edu
>>Subject: Cloning trick
>>
>>Dear All,

>>I would to ask if there is anybody mastered in working with restriction a=
>>nd digestion.
>>I want to clone a pcr fragment inside a binary vector. To do it efficient=
>>ly, I want to use a restriction enzyme after ligating vector and fragment=

>>, which cuts the original uncut vector before transformation.
>>what method do you offer?
>>Best regards,
>>Nasser.
>>
>>_______________________________________________
>>Methods mailing list
>>Met...@net.bio.net
>>http://www.bio.net/biomail/listinfo/methods
>>
>>

>>This email message may contain legally privileged and/or confidential inf=
>>ormation.  If you are not the intended recipient(s), or the employee or a=
>>gent responsible for the delivery of this message to the intended recipie=
>>nt(s), you are hereby notified that any disclosure, copying, distribution=
>>, or use of this email message is prohibited.  If you have received this =
>>message in error, please notify the sender immediately by e-mail and dele=

>>te this email message from your computer. Thank you.

>>--_002_AE06C4610E75404183F550518E585DBC264B821D17MSXMBCCR2rosw_--

>>
> _______________________________________________
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David Minde MSc (TUM)

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