Team:Duke/Notebook/MayJun

From 2014.igem.org

(Difference between revisions)
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</div>
</div>
 +
<div class="day">
 +
<a id="jun4"><h2>June 4</h2></a>
 +
<div class="obj">Objective: Prepare buffers and mediums for new CCEC protocol</div>
 +
<div class="ppl">Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper</div>
 +
<div class="lab">
 +
<p> Prepare SOB Medium for bacterial transformation</p>
 +
<ul>
 +
<li>Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells</li>
 +
<li>Made 1 L, autoclaved and stored in two 500 mL containers in cold room</li>
 +
<ul>
 +
<li>medium still appeared cloudy before autoclave--may just be new recipe</li>
 +
</ul>
 +
</ul>
 +
<p> Prepare CCMB 80 Buffer for making chemically competent E. coli cells</p>
 +
<ul>
 +
<li>Protocol from iGEM’s <a href="http://parts.igem.org/Help:Protocols/Competent_Cells">parts website </a></li>
 +
<li>Made 1 L, filtered and stored in two 500 mL containers in cold room</li>
 +
<ul>
 +
<li>pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed</li>
 +
</ul>
 +
</ul>
-
6/4/14
+
<p> Autoclave two 500 mL culture flasks</p>
 +
<ul>
 +
<li>For CCEC protocol</li>
 +
<li>With water inside to remove detergent residues</li>
 +
</ul>
-
Objective: Prepare buffers and mediums for new CCEC protocol
+
<p> Next steps: </p>
-
Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper
+
<ul>
-
Prepare SOB Medium for bacterial transformation
+
<li>Prepare CCEC</li>
-
Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
+
</ul>
-
Made 1 L, autoclaved and stored in two 500 mL containers in cold room
+
-
medium still appeared cloudy before autoclave--may just be new recipe
+
-
Prepare CCMB 80 Buffer for making chemically competent E. coli cells
+
-
Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
+
-
Made 1 L, filtered and stored in two 500 mL containers in cold room
+
-
pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed
+
-
Autoclave two 500 mL culture flasks
+
-
For CCEC protocol
+
-
With water inside to remove detergent residues
+
-
Next steps:
+
-
Prepare CCEC
+
-
Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC
+
<div class="obj">Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC</div>
-
Matthew Faw
+
<div class="ppl">Matthew Faw</div>
-
The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed.  Today, we are trying to see if we can get any transformed cells to grow in plates.   
+
<p> The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed.  Today, we are trying to see if we can get any transformed cells to grow in plates.  </p>
-
Followed Charlie’s Cloning protocols,with slight modifications
+
<ul>
-
Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure
+
<li>Followed Charlie’s Cloning protocols,with slight modifications</li>
-
Plated the transformed DNA on 2 separate plates, put in put in 37C overnight
+
<ul>
-
Results: No colonies grew (6/5/14)
+
<li>Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure</li>
-
Next Steps:
+
</ul>
-
-Examine plates to see if any cultures grew
+
<li>Plated the transformed DNA on 2 separate plates, put in put in 37C overnight</li>
-
-Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here: http://parts.igem.org/Help:Protocols/Competent_Cells
+
<li>Results: No colonies grew (6/5/14)</li>
-
-Lab currently in the process of making these CCEC
+
</ul>
-
Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped
+
<p> Next Steps:</p>
-
Matthew Faw, Charlie Cooper
+
<ul>
-
Prepare pdCas9 and pCsy4 to be miniprepped tomorrow
+
<li>Examine plates to see if any cultures grew</li>
-
Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol
+
<li>Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found <a href="http://parts.igem.org/Help:Protocols/Competent_Cells">here</a>: </li>
-
Put pCsy4 in a culture tube with 5ml SOC+Amp
+
<li>Lab currently in the process of making these CCEC</li>
-
Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them
+
</ul>
-
Next steps:
+
<div class="obj">Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped </div>
-
Miniprep plasmid DNA
+
<div class="ppl">Matthew Faw, Charlie Cooper</div>
 +
 
 +
<p> Prepare pdCas9 and pCsy4 to be miniprepped tomorrow</p>
 +
<ul>
 +
<li>Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol</li>
 +
<li>Put pCsy4 in a culture tube with 5ml SOC+Amp</li>
 +
<li>Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them</li>
 +
</ul>
 +
 
 +
<p>Next steps:</p>
 +
<ul>
 +
<li>Miniprep plasmid DNA </li>
 +
</ul>
 +
</div>
 +
</div>

Revision as of 20:33, 29 July 2014

May 2014
Month 2 of Project
sun mon tue wed thu fri sat
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

June 2014
Month 3 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30

May 29
Objective: Prepare Chemically competent E. Coli
Matthew Faw

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

  • Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

  • Transformation

June 1
Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

  • Followed Charlie’s Cloning Protocols
    • Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
  • Plated the transformed DNA and put in incubator at 37C
  • Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

  • Look at plates, compare cultures

June 2
Objective: Redo yesterday’s transformation
Matthew Faw

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

  • Followed Charlie’s Cloning Protocols
    • Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
  • Plated the transformed DNA on 6 separate plates, put in 37C overnight
  • Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

  • Look at plates, compare cultures, etc.

June 4
Objective: Prepare buffers and mediums for new CCEC protocol
Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper

Prepare SOB Medium for bacterial transformation

  • Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
  • Made 1 L, autoclaved and stored in two 500 mL containers in cold room
    • medium still appeared cloudy before autoclave--may just be new recipe

Prepare CCMB 80 Buffer for making chemically competent E. coli cells

  • Protocol from iGEM’s parts website
  • Made 1 L, filtered and stored in two 500 mL containers in cold room
    • pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed

Autoclave two 500 mL culture flasks

  • For CCEC protocol
  • With water inside to remove detergent residues

Next steps:

  • Prepare CCEC
Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC
Matthew Faw

The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates.

  • Followed Charlie’s Cloning protocols,with slight modifications
    • Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure
  • Plated the transformed DNA on 2 separate plates, put in put in 37C overnight
  • Results: No colonies grew (6/5/14)

Next Steps:

  • Examine plates to see if any cultures grew
  • Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here:
  • Lab currently in the process of making these CCEC
Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped
Matthew Faw, Charlie Cooper

Prepare pdCas9 and pCsy4 to be miniprepped tomorrow

  • Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol
  • Put pCsy4 in a culture tube with 5ml SOC+Amp
  • Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them

Next steps:

  • Miniprep plasmid DNA