Use of restriction enzymes

Tue Sep 4 17:49:07 BRT 2001

Dear Giovanni,
Thank you for your suggestions. In fact I tried  larger volume of the digestion reaction
with higher RE concentration and used less PCR product as suggested by Promega technical
service and it worked. I am trying now to use less volume to save on the RE but at least I
know that the PCR product has the restriction site. Thanks again
Laila

Giovanni Widmer wrote:

> Dear Laila,
> The fact that a DNA fragment has been amplified from Leishmania or another organism,
> will not affect if it's cut or not by a specific restriction enzyme. Since HaeIII is a
> 4-base cutter, any plasmid will carry multiple restriction sites. Take for instance
> some pUC or pBR plasmid DNA and digest it per manufacturer's recommendation, run it on
> an agarose gel side-by-side with uncut plasmid and you should see a clear difference.
> If, using the same conditions, your PCR product still doesn't cut, there is probably
> no HaeIII site in your PCR product - there might be a novel mutation, or your PCR
> product originates from a different locus.
> Good luck.
> Giovanni Widmer
> Tufts Univeristy School of Veterinary Medicine
> North Grafton, Massachusetts
>
> "Dr. Laila Nimri" wrote:
>
> > Dear colleagues,
> > I am using the restriction enzyme (Hae III, from Promega) to digest a PCR product
> > of  Leishmania DNA for the purpose of characterization of these spp. I did the
> > digest several times according to the manufacturer's instructions with no luck. I
> > also increased the enzyme concentration but it didn't work, I only got a band of
> > the undigested DNA. I would really appreciate it if anyone who used this
> > restriction enzyme can provide me with the right conditions to use. It would be
> > nice if I could also have the optimum conditions of other restriction enzymes that
> > worked on Leishmania.
> > Laila Nimri
> >
> > Jeffrey Shaw wrote:
> >
> > > Dear Colleagues,
> > > As some of you know, I am constructing a comprehensive database of sand
> > > flies in colony throughout the world. So far the response has been very
> > > good and I now have 56 colonies of 27 species registered in the database.
> > > Many thanks to those who have contributed so far. Now I would like to
> > > broaden the search for other existing colonies, particularly those in
> > > Europe. If my memory serves me correctly, there was an address list (e-mail
> > > list) generated at the ISOPS III in Montpelier of all attendees. If any of
> > > you can help me obtain a copy of that list it will be greatly appreciated.
> > > I'm sure Micheli Maroli knows of some colonies in Italy, but I don't have
> > > his e-mail address either. Do you?
> > > I ask for your help in disseminating this request for information about
> > > existing sand fly colonies? If you know of other workers who have colonies,
> > > please forward this message to them or send me their e-mail or mailing
> > > addresses and I will contact them directly.
> > > The essential elements of information needed on each colony are:
> > > 1. Name of Laboratory
> > > 2. Investigator Name(s)
> > > 3. Complete mailing address(es) of investigator(s)
> > > 4. Phone number (include country and city codes)
> > > 5. Fax number (include country and city codes)
> > > 6. E-mail address(es) of investigator(s)
> > > 7. Species in colony (list all strains of each species in colony)
> > > 8. Site of origin (for each species and strain)
> > > 9. Approximate number of generations in colony
> > > 10. Larval diet used
> > > 11. Adult diet used (human, hamster, rabbit, membrane, etc.;type of sugar
> > > meal)
> > > 12. Rearing temperature and relative humidity
> > > 13. Light Dark Cycle (12light/12dark; 16 light/8 dark)
> > > 14. Associated references (publications about the colony or publications
> > > resulting from experiments conducted using colony material)
> > > This database is intended to be a resource for all sand fly workers and will
> > > be made available on the web. It will be updated from time to time as more
> > > information is received.
> > > Thank you sincerely,
> > > Phillip G. Lawyer, Ph.D.
> > > Uniformed Services University of the Health Sciences
> > > 4301 Jones Bridge Road
> > > Bethesda, MD 20814, USA
> > > Phone: (301) 295-3735
> > > Fax: (301) 295-3860
> > > E-mail: <plawyer at usuhs.mil>
> > >
> > > --MS_Mac_OE_3067232602_15238671_MIME_Part
> > > Content-type: text/html; charset="US-ASCII"
> > > Content-transfer-encoding: quoted-printable
> > > Dear Colleagues,
> > >
> > > As some of you know, I am constructing a comprehensive database of sand
> > > fli= es in colony throughout the world. So far the response has been very
> > > g= ood and I now have 56 colonies of 27 species registered in the database.
> > > &nb= sp;Many thanks to those who have contributed so far. Now I would like
> > > = to broaden the search for other existing colonies, particularly those in
> > > Eur= ope. If my memory serves me correctly, there was an address list
> > > (e-ma= il list) generated at the ISOPS III in Montpelier of all attendees.
> > > If= any of you can help me obtain a copy of that list it will be greatly
> > > apprec= iated. I'm sure Micheli Maroli knows of some colonies in Italy, but
> > > I don't = have his e-mail address either. Do you?
> > >
> > > I ask for your help in disseminating this request for information about
> > > exi= sting sand fly colonies? If you know of other workers who have
> > > colonie= s, please forward this message to them or send me their e-mail or
> > > mailing ad= dresses and I will contact them directly.
> > >
> > > The essential elements of information needed on each colony are:
> > >
> > > 1. Name of Laboratory
> > > 2. Investigator Name(s)
> > > 3. Complete mailing address(es) of investigator(s)
> > > 4. Phone number (include country and city codes)
> > > 5. Fax number (include country and city codes)
> > > 6. E-mail address(es) of investigator(s)
> > > 7. Species in colony (list all strains of each species in colony)
> > > 8. Site of origin (for each species and strain)
> > > 9. Approximate number of generations in colony
> > > 10. Larval diet used
> > > 11. Adult diet used (human, hamster, rabbit, membrane, etc.;type of s= ugar
> > > meal)
> > > 12. Rearing temperature and relative humidity
> > > 13. Light Dark Cycle (12light/12dark; 16 light/8 dark)
> > > 14. Associated references (publications about the colony or publicati= ons
> > > resulting from experiments conducted using colony material)
> > >
> > > This database is intended to be a resource for all sand fly workers and wil= l
> > > be made available on the web. It will be updated from time to time as= more
> > > information is received.
> > >
> > > Thank you sincerely,
> > >
> > > Phillip G. Lawyer, Ph.D.
> > > Uniformed Services University of the Health Sciences
> > > 4301 Jones Bridge Road
> > > Bethesda, MD 20814, USA
> > >
> > > Phone: (301) 295-3735
> > > Fax: (301) 295-3860
> > > E-mail: <plawyer at usuhs.mil> = ;
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