haunr at nih.gov
Wed Sep 7 14:24:03 EST 1994
In article <lawa-0609941408470001 at pc0519.ri.afrc.ac.uk> lawa at bbsrc.ac.uk (Andy Law \(Big Nose\)) writes:
>From: lawa at bbsrc.ac.uk (Andy Law \(Big Nose\))
>Subject: Re: Subcloning PCR
>Date: 6 Sep 1994 14:26:59 -0500
>Organization: Roslin Institute
>Sender: nobody at cs.utexas.edu
>Message-ID: <lawa-0609941408470001 at pc0519.ri.afrc.ac.uk>
>References: <199409020012.RAA17831 at netcomsv.netcom.com>
>In article <199409020012.RAA17831 at netcomsv.netcom.com>,
>jloring at genpharm.com ("Jeanne Loring") wrote:
>> Hello netters- I followed some of the recent thread about the problems with
>> cloning PCR fragments, and, since I now have the problem, I wish I'd listened
>> more carefully. I cut my fragment with the restriction enzymes whose
>> believed I had included (with a 5'clamp), but there was no way to be sure they
>> both worked (SacI and XhoI). Now I'm not getting inserts into
>Bluescript II. A
>> colleague is having a similar problem with an entirely different sequence and
>> plasmid. Was there a summary on the net about solutions? I did read that
>> treatment of the Taq-produced fragment with the Klenow fragment of DNA
>> polymerase solved the problem for some people. Has anyone else had luck with
>> that, or can you suggest another solution?
>Basically IMHO there are two possible solutions that have worked for me.
>1) Forget about the fancy restriction-sites-in-my-primers method.
>Sacrifice directional cloning and use a T-tailed vector to directly clone
>the PCR product.
>2) Digest the PCR product with Proteinase K PRIOR to restriction digestion
>(Crowe et al., NAR 19)
>Doing both of the above i.e. digesting with PK prior to direct ligation
>into a T-tailed vector covers all eventualities. I found that 1) alone was
>successful nearly 100% of the time.
>If you do want the direction restriction site method, then remember to
>include at least 3 bases (in my experience) hanging over the end of the
>restriction site. Primers with sites right at the end of their sequence DO
>NOT WORK. If your primers are designed like this (with the restriction
>site right at the end) then don't panic. Just clone it into a T-tailed
>vector, grow it up, cut it out of the plasmid and then clone it in the
>direction you want.
>( Lawa @ bbsrc.ac.uk Big Nose in Edinburgh )
If you do not want to sacrifice directional cloning, but you are not having
success digesting your PCR products containing restriction sites at the ends
you may want to consider trying a ligation-independent vector. Briefly,
these vectors provide non-complementary, extended single-stranded tails after
treatment with T4 DNA polymerase. PCR products produced with primers
containing 5'-terminal sequences complementary to the ss tails of the vector
can be annealed (after appropriate T4 DNA pol treatment) to the vector and
transformed directly without ligation.
The advantages of this type of cloning are:
1. The PCR product can be directionally cloned.
2. The non-complementary tails of the vector produce very low background.
3. You do not have to digest the PCR product with a restriction enzyme,
therefore, you do not have to worry about internal restriction sites.
4. No ligation step is required, so the cloning procedure is accelerated.
5. The cloning vectors come in several different flavors (commercially
prepared vectors can be obtained from Clontech or PharMingen).
The major disadvantage of this type of cloning is:
1. Primers used for PCR must contain 5'-terminal extensions of ~12 nt.
However, if you are currently adding restriction sites to your primers (let's
say 6 nt) and you now have to add a 'clamp' at the 5' side of the restriction
site (3-8 nt), you are approaching (or exceeding) the required extensions for
ligation-independent cloning. And the efficiency of cloning by this method
may offset any additional costs incurred by using somewhat longer primers.
If directional cloning is not a requirement, then the TA-vectors work great.
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