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