IGEM:MIT/2006/Notebook/2006-6-13

06/13/06 Lab

 * 1) Made glycerols of BAMT and SAMT from BL21 (6/9/06)
 * 2) *Made two copies (4 total) of each by adding 1ml of each to glycerol stock (let sit for 30 min)
 * 3) Created minipreps of the BAMT and SAMT from BL21 (6/9/06)
 * 4) *Used the protocol outlined: http://openwetware.org/wiki/Miniprep/Qiagen_kit
 * 5) *Spun total of 3.6 mls of each- [as not many cells in culture, added more (to 3.6) and spun again]
 * 6) *Added 250µl of P1 and vortexed, added 250µl of P2 and inverted a couple of times, added 350µl of N3 and centrifuged for 10 min
 * 7) *Washed with PB (optional in protocol, but done here)
 * 8) *Added only 35µl instaed of 50µl of EB (DNA may be on low side)
 * 9) Created additional liquid cultures of colonies from Top10(TK) 200µl plates
 * 10) *3 from each of SAMT, BAMT, and BSMT from 6/12/06.
 * 11) *Added 10mL of LB for liquid cultures


 * RS 14:15, 13 June 2006 (EDT): Just after you left we realized that no one had started BL21DE3 cultures (without plasmid) as a negative control for tomorrow. So I went ahead and took some competent cells and streaked a plate and started a 100mL culture for you.  This will serve as a good point of comparison when "testing" the cultures tomorrow.

pET-28 Primers

 * 1) T7 (TTA ATA CGA CTC ACT ATA GGG) (T7 promoter)
 * 2) TphiBBAP (CCT TCT GCA GCG GCC GCT ACT AGT ACA AAA AAC CCC TCA AGA CCC GTT TAG AGG CC CAA GGG) [T7 terminator]

Sequencing

 * pET28a(+) genbank file from Novagen <- Is this is the vector that the constructs came in?
 * This vector has a T7 promoter primer binding site but no T3, SP6, M13 Forward or M13 Reverse primer binding site.
 * Does anyone have a T7 terminator primer?