66 -- CONSTRUCT, PLATE, PICK AND GRID A BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY USING MALE RAINBOW TROUT GENOME

Notice Date

March 28, 2001

Contracting Office

USDA, ARS, North Atlantic Area, Contracting Office, 600 East Mermaid Lane, Wyndmoor, PA 19038

ZIP Code

19038

Solicitation Number

03-3615-01

Response Due

April 3, 2001

Point of Contact

Lisa Botella, Point of Contact, (215) 233-6551, fax (215) 233-6558, Eileen LeGates, Contracting Officer

Description

Correction: The requirement was initially synopsized on March 7, 2001. Amendment No. 01 was issued on March 14, 2001. Amendment No. 02 was issued on March 28, 2001. This amendment provides: 1) Answers to questions received in response to the subject solicitation: QUESTION: In case you do not know, the distribution of insert sizes of a BAC library looks like a bell shaped curve. Unfortunately, it is technically impossible to make a BAC library with ZERO clones having inserts grater than 150,000 bp and ZERO clones having inserts less than 100,000 bp with an average insert size of 135,000 bp. There will be a small percentage of BAC clones outside this range. ANSWER: Revise Technical Specification A .5 (3) to: The BAC library shall have an average insert size of 135,000 base pairs, with 90% of the inserts falling between the ranges of 100,000 base pairs and 150,000 base pairs. Revises and replace page 6 in it's entirety. Question: The last line in Section A.5(3) "shall be selected for ligation into the vector. This is not what happens. When one is building a BAC library, the protocol is to perform two unique size selection on partially digested genomic DNA to obtain fragments for the ligation. The size range that is "selected for ligation into the vector" is from four to six Pulsed Field Gel "DNA zones" that are between 300,000 bp and 1,000,000 bp (not the size range for which the final inserts will be)! Then these "DNA zones" are all tested (ligate DNA into vector and transform and DNA is prepped from random BAC clones which are then sized on a Pulsed Field Gel). The "DNA zone" which gives the appropriate insert size range and has a high number of transformants is used to make the hundreds of transformations required to build the library. Would you consider revising Section A.5.(3) to include a percentage of inserts between the required ranges? ANSWER: See response to previous question. 2) THE HOUR AND DATE SPECIFIED FOR RECEIPT OF OFFERS HAS BEEN CHANGED TO 4:00 P.M. (LOCAL TIME) ON APRIL 3, 2001. All other information in the initial synopsis is correct.

Record

Loren Data Corp. 20010330/66SOL014.HTM (W-087 SN50H5Q6)