Handling Other Peoples Clones

 

CLONE DATA:

1. Copy the map and letter and place in Pam’s file and lab file.

Record the vector, insert size and full polylinker region

3. Pull down vector sequence and insert sequence from NCBI http://www.ncbi.nlm.nih.gov/

Choose search Genbank and fill in clone name

Choose full length CDS and record the accession number

4. Create map in Strider by pasting in the genbank sequence. Make sure that the coding region is in upper case and non-coding is in lower case. Annotate important clone information in the box below.

HANDLING THE CLONE

1. Soak paper containing clone in 50-100ul of sterilized and filtered TE

2. Transform 2 ul and 10 ul into JM109

Give the rest to Pam to be stored frozen in the original clone box.

3. Grow up miniprep and plasmid prep.

Give 200ug CsCl double-banded DNA to Pam for the clone store.

4. Map clone fully by running 1ug uncut and 1ug digested with each enzyme in the polylinker.

5. Sequence the ends if necessary.

Sequences from the facility can be opened in MS word and pasted into BLAST

Align sequence using BLAST search http://www.ncbi.nlm.nih.gov/BLAST/

Use BLAST 2 sequences choice

Feed the accession # of known seq in seq1 box and seq from facility in seq2 box.

6. Create fully annotated final strider map.