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

06/13/06 Lab

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

Notes

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?