Team:Hawaii/Notebook/2008-07-22

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(New page: {{Team:Hawaii/Header}} = Things we did today = == Wetlab work == ===Alkaline Lysis Mini-prep=== :<strong> Margaret</strong> :* Plasmid prep of pSB1A2, pSB1A3, and pSB3K3: 3 1.5 mL preps o...)
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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.
 +
:*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>
+
:* nir, pilA, slr-1, slr-2, GFP fusion
 +
:* Added 1 &mu;l 10mg/ml RNAse to each plasmid prep after transferring supernatant; incubated at 55C for 90 min.
===Glycerol Stock===
===Glycerol Stock===
Line 15: Line 17:
:<strong>Margaret</strong>
:<strong>Margaret</strong>
-
:*glycerol stock of pSB1A2, pSB1A3, and pSB3K3
+
:*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.
:<strong>Grace</strong>
:<strong>Grace</strong>
 +
:* nir, pilA, slr-1, slr-2, GFP fusion, BB-pRL1383a, BBa_E0026
===pRL1383a insert Clean-up===
===pRL1383a insert Clean-up===
-
 
+
:<strong>Margaret</strong>
:*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
:*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.
:*Cleaned the oriT construct using the Qiagen Minelute Gel Purification kit.
-
== Drylab Work ==
+
===Made 80% glycerol stock solution===
 +
:<strong>Grace </strong>
 +
 
 +
===Organized plasmid preps===
 +
:<strong>Grace </strong>
 +
 
 +
:* 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
-
===Name of Task===
+
===BioBrick assembly/subcloning ===
 +
:<strong>Grace </strong>
 +
:* Extracted GFP, B0024, B0034 from gel using MiniElute Spin Kit from Qiagen
 +
:* Checked concentration of parts using nanodrop
 +
<div style="text-align: center;">'''DNA concentration of parts extracted from agarose gel'''</div>
 +
{|align="center" border="1"
 +
! Part
 +
! Concentration
 +
|-
 +
|align="center"|BBa_B0024
 +
|align="center"|15.5 ng/&mu;l
 +
|-
 +
|align="center"|BBa_B0034
 +
|align="center"|2.2 ng/&mu;l
 +
|-
 +
|align="center"|GFP
 +
|align="center"|5.2 ng/&mu;l
 +
|-
 +
|align="center"|GFP fusion (from 7/15/08 gel purification)
 +
|align="center"| 1.0 ng/&mu;l
 +
|-
 +
|align="center"|''nir'' promoter (from 7/11/08 gel purification)
 +
|align="center"|10.9 ng/&mu;l
 +
|}
 +
:* Ligated parts
 +
::* nir + B0034 (rbs)
 +
::* GFP + B0024 (tt)
 +
::* GFP fusion + B0024 (tt)
 +
::* Incubated ligation reaction for 2 hours at room temperature
 +
:* Transformed DH5&alpha; with ligated parts
 +
::* Used 5 &mu;l ligation reaction for each transformation
= Discussion =
= Discussion =

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


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