No subject

phenol-chloroform with isoamyl alchohol and to avoid sand fly pooling. 

Good luck
Omar Hamarsheh,Msc., PhD


-------------------------------------
Omar Hamarsheh, MSc., PhD
Al-Quds University
Department of Biological Sciences
P.O.Box 51000
Jerusalem
Via ISRAEL

Tel: 00972-599197097
Fax: 00972-22796960

Email: ohamarsheh at gmail.com
          mail.alquds.edu/~f3706/


-----Original Message-----
From: leish-l-bounces at lineu.icb.usp.br
[mailto:leish-l-bounces at lineu.icb.usp.br] On Behalf Of Hanafi, Hanafi CTR EG
NAMRU3 
Sent: Tuesday, July 12, 2011 12:17 PM
To: Leish-L
Cc: Obenauer, Peter J. LCDR; Kaldas, Rania M CIV EG NAMRU3
Subject: [Leish-l] PCR and Leish detection in sand flies

Dear Leish-L

Does anyone can help explain what happened to our PCR run to test some sand
flies for Leish.

We tested 3 pools of SF collected from endemic CL foci in Libya. Details
were as follow:

1- DNA extraction using Qiagen DNA mini Kit
2- Amplification using Leishmania GENUS primer/probe set
3- Ct for 3 pools were: A) P. papatasi (Ct: 36.0621)
                        B) P. papatasi (Ct: 36.046)
                        C) P. longicuspis (Ct: 35.7944)

4- Amplification using Internal Transcribed spacer 1 primers according to
Schonian 2003 (L5.8S and LITS.R) showed faint bands.
5- Reamplified ITS1 region using the same primers and PCR conditions, and by
running the samples on 3% Agarose gel, is gave a smeary band that stuck in
the well, didn't run like the controls.
6- Step 4 & 5 were repeated but the same outcome appeared.
7- Using Leishmania major specific primer/probe sets, samples were NEGATIVE
for L. major, controls were excellent.
8- Tried Nested ITS1 according to Schonian 2003, using the same primers and
PCR conditions.
9- Same smeary gel appeared again and we don't know why. Please advise

Thank you

Regards
Hanafi

Hanafi  A.  Hanafi, Ph. D.
Medical Research Scientist
Vector Biology Research Program
U.S. Naval Medical Research Unit No.3
Cairo, Egypt
Tel & Fax:  ++2-02-2342-3090
E-mail: hanafi.hanafi.ctr.eg at med.navy.mil
        yahanafi at yahoo.com