Brenda,
I constructed the hairpin using two fragments differing in length by 113 bp using the primers with synthetic restriction sites BamHI/ SalI and SphI/SalI . The PCR amplified fragments were restricted with SalI and  ligated to each other in reverse orientation. For Ligation ,I used Takara Mighty mix (16oC in thermocycler for 30 minutes). The ligated fragment was cloned into BglII/SphI- digested pMB38 vector  using Takara mighty mix.
If you have more questions or need the protocol, let me know.
Regards,
Sandhya.


Dr. Sandhya N Baviskar
Assistant Professor
Department of Biology
University of Arkansas- Fort Smith
5210 Grand Ave.
Fort Smith AR 72913
Phone : 479-788-7789
________________________________________
From: DICTY [[log in to unmask]] On Behalf Of Brenda Blacklock [[log in to unmask]]
Sent: Wednesday, May 19, 2010 3:11 PM
To: [log in to unmask]
Subject: [DICTY - 331] long hairpin RNAi construct

Hello All,

We are trying to construct a long hairpin for RNAi using essentially the
technique described by Rosel and Kimmel for COP9.  We have the long arm
of the hairpin (1100 bp) in pMB38 but are having problems inserting the
short arm (800 bp).  Does anyone have any tricks for getting what seems
to be a difficult ligation to work?

Thanks for any suggestions you may have for us.

Brenda Blacklock