Team:Hawaii/Notebook/2008-07-22

From 2008.igem.org

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(Alkaline Lysis Mini-prep)
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:* 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.
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:*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

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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


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