IGEM:MIT/2006/Notebook/2006-6-13: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 5: | Line 5: | ||
#*Used the protocol outlined: http://openwetware.org/wiki/Miniprep/Qiagen_kit | #*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] | #*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 250µl of P1 and vortexed, added 250µl of P2 and inverted a couple of times, added 350µl of N3 and centrifuged for 10 min | ||
#Created additional liquid cultures of colonies from Top10(TK) 200µl plates | #Created additional liquid cultures of colonies from Top10(TK) 200µl plates | ||
#*3 from each of SAMT, BAMT, and BSMT from 6/12/06. | #*3 from each of SAMT, BAMT, and BSMT from 6/12/06. | ||
#* | #*Added 10mL of LB for liquid cultures | ||
#*Washed with PB (optional in protocol, but done here) | |||
==To do== | ==To do== |
Revision as of 09:11, 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
- 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
- Washed with PB (optional in protocol, but done here)
To do
- Miniprep two colonies of each construct
- Submit for sequencing with T7 forward and reverse primers (need to find primers).
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?