Use of restriction enzymes

[Giovanni Widmer]

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> = ;