IGEM:MIT/2006/Notebook/2006-6-13: Difference between revisions
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#*Added 10mL of LB for liquid cultures | #*Added 10mL of LB for liquid cultures | ||
== | ==pET-28 Primers== | ||
# | #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== | ==Notes== |
Revision as of 10:15, 13 June 2006
06/13/06 Lab
- 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
pET-28 Primers
- 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?