"
Team:Hawaii/Notebook/2008-07-22
From 2008.igem.org
(Difference between revisions)
(→Alkaline Lysis Mini-prep) |
m |
||
| Line 7: | Line 7: | ||
:* Plasmid prep of pSB1A2, pSB1A3, and pSB3K3: 3 1.5 mL preps of the first two, and 10 of the third because it has a lower copy number. Used the protocol from Dr. Callahan's lab ([[http://packrat.stjohn.hawaii.edu/prestinglab/wiki/Protocols]]). | :* Plasmid prep of pSB1A2, pSB1A3, and pSB3K3: 3 1.5 mL preps of the first two, and 10 of the third because it has a lower copy number. Used the protocol from Dr. Callahan's lab ([[http://packrat.stjohn.hawaii.edu/prestinglab/wiki/Protocols]]). | ||
:* I had to skip the phenol/chloroform step, so after adding Rnase A and incubating for 20 minutes, I spun down again, collected the supernatant and then precipitated with isopropanol. | :* I had to skip the phenol/chloroform step, so after adding Rnase A and incubating for 20 minutes, I spun down again, collected the supernatant and then precipitated with isopropanol. | ||
| + | :*The write-up for this prep can be found [[Team:Hawaii/Plasmid Prep|here]] under the 7/23 attempt. | ||
:<strong>Grace </strong> | :<strong>Grace </strong> | ||
Latest revision as of 00:40, 26 July 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Alkaline Lysis Mini-prep
- Margaret
- Plasmid prep of pSB1A2, pSB1A3, and pSB3K3: 3 1.5 mL preps of the first two, and 10 of the third because it has a lower copy number. Used the protocol from Dr. Callahan's lab (http://packrat.stjohn.hawaii.edu/prestinglab/wiki/Protocols).
- I had to skip the phenol/chloroform step, so after adding Rnase A and incubating for 20 minutes, I spun down again, collected the supernatant and then precipitated with isopropanol.
- The write-up for this prep can be found here under the 7/23 attempt.
- Grace
- nir, pilA, slr-1, slr-2, GFP fusion
- Added 1 μl 10mg/ml RNAse to each plasmid prep after transferring supernatant; incubated at 55C for 90 min.
Glycerol Stock
- Margaret
- glycerol stock of pSB1A2, pSB1A3, and pSB3K3. These DB3.1 strains containing these plasmids can be found in the -80C with the other plasmids in E. coli.
- Grace
- nir, pilA, slr-1, slr-2, GFP fusion, BB-pRL1383a, BBa_E0026
pRL1383a insert Clean-up
- Margaret
- in preparation for constructing the biobrick form of pRL1383a, cleaned the PCR products: mob, rep, oriV, and the aadA region using the "Isopropanol Precipitation for PCR purification" protocol from openwetware.org
- Cleaned the oriT construct using the Qiagen Minelute Gel Purification kit.
Made 80% glycerol stock solution
- Grace
Organized plasmid preps
- Grace
- Determined DNA concentrations of all plasmid preps to date
- Relabeled all tubes w/ plasmid (or BioBrick part), DNA concentration, date of prep, and initials of person who prepped
- Posted more detailed plasmid prep information on private website
BioBrick assembly/subcloning
- Grace
- Extracted GFP, B0024, B0034 from gel using MiniElute Spin Kit from Qiagen
- Checked concentration of parts using nanodrop
DNA concentration of parts extracted from agarose gel
| Part | Concentration |
|---|---|
| BBa_B0024 | 15.5 ng/μl |
| BBa_B0034 | 2.2 ng/μl |
| GFP | 5.2 ng/μl |
| GFP fusion (from 7/15/08 gel purification) | 1.0 ng/μl |
| nir promoter (from 7/11/08 gel purification) | 10.9 ng/μl |
- Ligated parts
- nir + B0034 (rbs)
- GFP + B0024 (tt)
- GFP fusion + B0024 (tt)
- Incubated ligation reaction for 2 hours at room temperature
- Transformed DH5α with ligated parts
- Used 5 μl ligation reaction for each transformation
Discussion
Quote of the Day
History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
