Team:Yale/Notebook/Week5

From 2011.igem.org

(Difference between revisions)
Line 23: Line 23:
</div>
</div>
<div id="entry">
<div id="entry">
-
 
+
<h1>Week 5: June 12-June 19</h1>
-
== Notebook: Week 5 ==
+
<ul>
-
 
+
<li>Monday:<ul>
-
''Monday''
+
<li>Ran another SDS-PAGE gel to verify expression, but unable to find protein (see Figure)</li>
-
 
+
Tuesday:<ul>
-
''Tuesday''
+
<li>Tried UV-vis 280 absorbance on Tokyo Tech eGFP-TmAFP fusion proteins; too messy (see lab notebook, Nanodrop pictures)</li>
-
Tried absorbance; too messy (see lab notebook, Nanodrop pictures)
+
<li>Fluorescence via fluorimetry of Tokyo Tech protein indicates GFP is found in supernatant, but not in pellet from lysate</li>
-
Fluorescence via fluorimeter indicates GFP is found in supernatant, but not in pellet from lysate
+
<li>Ran Western blots on ZeAFP and TmAFP from Tokyo Tech (see Figure)</li>
-
Tried running tris tricine gel instead of normal glycine gel
+
<li>Started planning Ice survival assays (need to do serial dilutions instead of changing the volume. After speaking to Fikrig lab, they told us this is better. If OD600 = 1, this is approximately 1e9 CFU/ml. If you do a dilution to 10^-5 and plate 100ul, you get 1000 colonies. 10^-6 you get 100 colonies, 10^-7 you get 10 colonies. You plate all these dilutions. No IPTG is needed on plates because after freezing you are done with the experiment. Media should be left overnight but better 2 days to cool at room temperature. Then store in fridge upside down. No fractures or moisture. Do to serial dilution use large volume,s 900ul diluant and 100ul of stuff. Can resuspend in media, or water, or PBS, or 0.9% NaCl solution, dpenending on how much want to stress. Repeat dilution series 2-3 times. Use new dilution series after frozen to replate dilution series). </li>
-
 
+
</ul></li><li>
-
''Wednesday''
+
Wednesday:<ul>
-
Protocols for sequencing (http://medicine.yale.edu/keck/dna/protocols/tube/index.aspx):
+
<li>Received Israel plasmids for variants of TmAFP</li>
-
 
+
<li>Transformed all Israel plasmids into DH5alpha</li>
-
- 500-600 ng ds plasmid DNA template
+
<li>Prepared sequencing samples to be sent to Keck Biotechnology Center</li>
-
 
+
<li>Started growing Israel plasmid transformed samples to OD 0.6, add 0.5mM IPTG, induce for 48 hours at 15C. After shaking centrifuge 30 min 2700g, 4C. Flash freeze store -20oC. Same with Canada GFP.</li>
-
- 2 microliters of 4 micromolar primer
+
</ul></li><li>
-
 
+
Thursday:<ul>
-
- fill up to 18 microliters water
+
<li>Received IDT Synthetic RiAFP gene: Took tube that we got from IDT, centrifuged to get DNA at bottom, resuspended in 20uL of 10mM Tris, 0.1mM EDTA buffer PH 7.5-8.0, to get 0.1ug/ul. Incubated rt 30 mins, vortexed 20s. Then centrifuged 1 min. Added 1ul of stock to 999ul water. Then used 2ul of this to xform bacteria. </li>
-
 
+
<li>PCR amplified ZeAFP, confirmed with agarose gel, then digested with XbaI and SpeI and ligated with T4 DNA ligase with BioBrick backbone</li>
-
For today, here are the amounts:
+
</ul></li><li>
-
 
+
</ul>
-
- MBP-His-TEV-AFP Starting Concentration: 135.8 ng/microliter
+
-
 
+
-
- malF: 124.7 ng/microliter
+
-
 
+
-
- malR: 107.1 ng/microliter
+
-
 
+
-
Use 4 microliters of MBP-His-TEV-AFP, which brings us to approximately 550 ng, which is the required amount of plasmid, add 4 microliters to each of two sequencing tubes. Next, I calculated molar concentration of our primers, via http://www.genscript.com/conversion.html:
+
-
 
+
-
In 1 microliter of malF, we have 124.7 ng, so to calculate the molarity: 124.7 ng*(10^3 pg/1 ng)*(1 pmol/330 pg)*(1/25 nucleotides) = 15.12 pmol in 1 microliter, or 15.12 micromolar malF; repeat similarly for malR to get 12.98 micromolar malR.
+
-
 
+
-
We need 4 micromolar primer, which I made a 10 microliter "stock" solution of, so the calculation is as follows: 15.12 micromolar*(x amount of primer) = 10 microliters*4 micromolar, so x = 2.64 microliters for malF and 3.08 microliters for malR.  I added that amount and then filled up to 10 microliters with water. I then took 2 microliters of each stock primer solution and added to the appropriate sequencing tube, then filled up to 18 microliters with water.  Finally, I labeled the tubes appropriately (http://medicine.yale.edu/keck/dna/protocols/tube/labeling.aspx).
+
-
 
+
-
 
+
-
http://128.36.30.107/fmi/iwp/cgi?-db=keck_dna_seq&-loadframes, go to 'Premix individual tube order'
+
</div>
</div>
<div style="clear:both"></div>
<div style="clear:both"></div>
</div>
</div>

Revision as of 23:19, 27 September 2011

iGEM Yale

Week 5: June 12-June 19

  • Monday:
    • Ran another SDS-PAGE gel to verify expression, but unable to find protein (see Figure)
      • Tuesday:
      • Tried UV-vis 280 absorbance on Tokyo Tech eGFP-TmAFP fusion proteins; too messy (see lab notebook, Nanodrop pictures)
      • Fluorescence via fluorimetry of Tokyo Tech protein indicates GFP is found in supernatant, but not in pellet from lysate
      • Ran Western blots on ZeAFP and TmAFP from Tokyo Tech (see Figure)
      • Started planning Ice survival assays (need to do serial dilutions instead of changing the volume. After speaking to Fikrig lab, they told us this is better. If OD600 = 1, this is approximately 1e9 CFU/ml. If you do a dilution to 10^-5 and plate 100ul, you get 1000 colonies. 10^-6 you get 100 colonies, 10^-7 you get 10 colonies. You plate all these dilutions. No IPTG is needed on plates because after freezing you are done with the experiment. Media should be left overnight but better 2 days to cool at room temperature. Then store in fridge upside down. No fractures or moisture. Do to serial dilution use large volume,s 900ul diluant and 100ul of stuff. Can resuspend in media, or water, or PBS, or 0.9% NaCl solution, dpenending on how much want to stress. Repeat dilution series 2-3 times. Use new dilution series after frozen to replate dilution series).
    • Wednesday:
      • Received Israel plasmids for variants of TmAFP
      • Transformed all Israel plasmids into DH5alpha
      • Prepared sequencing samples to be sent to Keck Biotechnology Center
      • Started growing Israel plasmid transformed samples to OD 0.6, add 0.5mM IPTG, induce for 48 hours at 15C. After shaking centrifuge 30 min 2700g, 4C. Flash freeze store -20oC. Same with Canada GFP.
    • Thursday:
      • Received IDT Synthetic RiAFP gene: Took tube that we got from IDT, centrifuged to get DNA at bottom, resuspended in 20uL of 10mM Tris, 0.1mM EDTA buffer PH 7.5-8.0, to get 0.1ug/ul. Incubated rt 30 mins, vortexed 20s. Then centrifuged 1 min. Added 1ul of stock to 999ul water. Then used 2ul of this to xform bacteria.
      • PCR amplified ZeAFP, confirmed with agarose gel, then digested with XbaI and SpeI and ligated with T4 DNA ligase with BioBrick backbone

Retrieved from "http://2011.igem.org/Team:Yale/Notebook/Week5"