Adding Restriction sites to Primers

choie01 at mcclb0.med.nyu.edu choie01 at mcclb0.med.nyu.edu
Thu Mar 10 01:23:17 EST 1994


In article <940309084116.2401704d at vax1.utulsa.edu>,
BMA14432 at vax1.utulsa.edu writes:

> Hello netters,
> I've picked a couple of primers to PCR my target and now wouuld like to
> add restriction sites to the 5' ends of them. What are the
> pitfalls, if any, to be avoided in doing this. Any helpful hints or
> anecdotes will be greatly appreciated! Thanks in advance

This is similar to a question that I had posted to this newsgroup
about a month or two ago.  I had received many helpful suggestions and
posted a summary of their replies.  Below, I've reposted my original
summary for you and for anyone else who may have missed it the first
time.

If there are any questions, you can e-mail me at the address below.  Once
again, thanks to all those who replied!

Elmer Choi
New York University Medical Center
Department of Pathology
e-mail:  choie01 at mcclb0.med.nyu.edu


BELOW IS A REPOSTING OF THE ORIGINAL SUMMARY

Dear Netters,

First of all, I would like to extend a hearty thanks to all of those
who took the time to reply to my query.

The consensus of the replies that I had received are listed below. If
anyone would like to add to this list, please feel free to e-mail me
or post it to this newsgroup.  Also, if I have misquoted anyone or
have misrepresented their statements, please accept my apology and
contact me as I will try to correct it immediately.

When engineering restriction sites into PCR primers...

1)  Check that the restriction sites chosen can be cut well at the
ends by that particular enzyme.

	brunstei at unixg.ubc.ca writes:
	In regards to potential problems with digestibility of the sites
one designs into the ends of PCR primers, I would reccomend checking out
the table "Cleavage close to the ends of DNA fragments" in the back of
NEB's catalog (p.182 in last year's edition, in case it's handy.)  I have
relied on this when picking my sites and found it quite useful

	Gil(KEINI at md2.huji.ac.il) writes:
	First you have to choose the sites. The enzyme site of your
choice should be easily cut when near the ends of DNA frag. Look in
the new-england-biolab catalog for "cleavage close to the end of DNA
fragments"-table (p.180-181 in 93/94 catalog). MAKE SURE THAT THE
SITES ARE DIFFERENT AND WON'T LIGATE TO EACH OTHER,


2)  Try adding a few extra bases 5' to the restriction site to "clamp"
down the end and allow the restriction enzyme to interact with the PCR
product.

	Barry (bjmarg at welchlink.welch.jhu.edu) writes:
	If you read Scharf's PCR book (the name of which escapes me,
but it was  published in 1990), there is a chapter on "Cloning through
PCR" in which  5' "clamps" are discussed.  You need to engineer these,
as well as the  restriction sites at the 5' ends.  Anyway... consult
the appendix in the  NEB catalog for appropriate clamp sequences; they
refer to them as  "cutting near the end of oligonucleatides."

3)  Gel purify your PCR product.

	Deborah A. Samac writes:
	The  critical part is to gel purify the pcr product away from
the other reaction  components in order to get good cutting and
ligation.
	[stuff in middle deleted]
Remove proteins, excess nucleotides and primers from DNA by running
sample out in a preparative minigel. I use 0.8% low melting agarose in
TAE. Tape 2 or 3 small wells (12 well comb) together and load all of
Klenow reaction into the megawell. Cut out band and purify using
GeneClean. I use 5 ul Glassmilk and elute in a total of 30 #L TE.

	brunstei at unixg.ubc.ca also writes:
	As far as purification goes, I always just isolate the PCR product
from the reaction directly by agarose gel electrophoresis and
electroelution of the band, it's quick and works at lest for me.


The only thing that I would like to add to this list is that if all
else fails in terms of cutting your PCR product and cloning into a
vector, TA clone the PCR product and then cut.  I have resorted to
this in the past in desperation.  It has worked fine for me in the
past...despite the added time.

Thanks again to all those who replied.

Elmer Choi
New York University Medical Center
Department of Pathology
email- choie01 at mcclb0.med.nyu.edu




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