Notebook:Tk/2008/04/08

Primers for recT gene

 * gtttcttcgaattcgcggccgcttctagatgaataataaaattgaattaatattaaataattc,recT-F
 * gtttcttctactagtagcggccgctgcagttattaatcagaaaacatttcattaacagc,recT-R
 * gaaatgcaagaagcctatcaacatgaccaagcaataattattaataacagc,recT-mut-F
 * gctgttattaataattattgcttggtcatgttgataggcttcttgcatttc,recT-mut-R

Vector NTI file for recT mutated gene

 * from S. citri protein CAK99381
 * mutated to eliminate GATC site
 * biobricked, entered as part J70007 []
 * [[media:recT-SC.gb]]

Transformations

 * transform E. coli at 2.0 KV outgrow in 1 ml SOC rotated
 * plate out 100 &mu;l at 1 hour/2.5 hour/3.5 hour
 * transform M. florum frozen cells
 * T1, perhaps sparked at 2.0 KV; outgrew anyway
 * T2, transformed at 2.0 KV without sparking
 * plated out 290 &mu;l at 1 hour and 2 hour and 3 hour
 * Added 1 ul Tet to 300 ul culture at 2 hours for T1 and 1 hour for T2
 * Plated out both tet and non-tet added cultures for T1 and T2

Made electrocompetent cells

 * 5 ml and 500 ul seed culture tubes (50 ml final) grown 18 hours at 30C
 * Spin down, resuspend in 45 ml EPB
 * again
 * again, in 20 ml
 * spin down, resuspend in remaining liquid, bring to 250 ul
 * add 35 ul 80% glycerol
 * Aliquot to 50 ul tubes
 * flash freeze in EtOH + dry ice