IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-10: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
mNo edit summary
No edit summary
Line 1: Line 1:
{{IGEM:/Harvard/2006/Cyanobacteria}}
<div class="tabs">
<ul>
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-9 | Previous Entry]]</li>
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-10 | Current Entry]]</li>
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-11 | Next Entry]]</li>
</ul>
</div>
<br style="clear:both">
<div class="tabcontent">
==Ligation Attempt #2 of J04450+PSB4A3 (see Monday)==
==Ligation Attempt #2 of J04450+PSB4A3 (see Monday)==
Previous ligation attempt did not survive the resistance innoculation. Due to the low amounts of the ligation product, will redo experiments but allign molar amounts as per Endy protocol instead of using a fixed amount like last time:
Previous ligation attempt did not survive the resistance innoculation. Due to the low amounts of the ligation product, will redo experiments but allign molar amounts as per Endy protocol instead of using a fixed amount like last time:


Although the Silver protocol recommends using 50-100ng BB vector + 4-10x insert molar mass, Endy recommends a 3-fold molar excess and less DNA. Roche kit says 1:1 or 1:2 works for DNA which is not similiar in length. Seeing as how our total vector + insert DNA is very low concentration, we will use a ''' 1:2 '''ratio.
Although the Silver protocol recommends using 50-100ng BB vector + 4-10x insert molar mass, Endy recommends a 3-fold molar excess and less DNA. Roche kit says 1:1 or 1:2 works for DNA which is not similiar in length. Seeing as how our total vector + insert DNA is very low concentration, we will use a ''' 1:2 '''ratio.

Revision as of 09:21, 10 August 2006

<html><style type='text/css'> .tabs { 

 font-size:80%;
 font-weight:none;
 width: 100%;
 color: #FFFFFF;
 background:#FFFFFF url("/images/5/54/DarkgreenTab-bg.gif") repeat-x bottom;

}

.tabs li { 

 background:url("/images/3/36/DarkgeenTab-left.gif") no-repeat left top;

}

.tabs a,.tabs strong { 

 background:url("/images/d/d3/DarkgreenTab-right.gif") no-repeat right top;
 color:#FFFFFF;
 padding: 3px 10px 3px 4px;

}

.tabs strong{ 

 color:#CCFF00;
 background-image:url("/images/b/b1/DarkgreenTab-right_on.gif");

}

.tabs a:hover{ 

 color:#66FF00;

}


</style></html>



Ligation Attempt #2 of J04450+PSB4A3 (see Monday)

Previous ligation attempt did not survive the resistance innoculation. Due to the low amounts of the ligation product, will redo experiments but allign molar amounts as per Endy protocol instead of using a fixed amount like last time:

Although the Silver protocol recommends using 50-100ng BB vector + 4-10x insert molar mass, Endy recommends a 3-fold molar excess and less DNA. Roche kit says 1:1 or 1:2 works for DNA which is not similiar in length. Seeing as how our total vector + insert DNA is very low concentration, we will use a 1:2 ratio.

Ligation

  • BB vector pSB4A3: 50ng @ BB backbone high copy 1 (3.2 ng/uL) = 15.625 uL
  • BB insert J04450: 2*(~1069bp/3339bp)*50ng=24.01 ng @ (6.0 ng/uL) = 5.33 uL
  • 1x DNA Dilution Buffer: 4 uL

Protocol (for this only because it is above 10uL):

  • Mix above ratios + 25uL #1 + 2.5uL #3
    • BE sure to mix #1 (ligation buffer) well especially!
  • Sit at bench for ~30min
  • Put back on ice

Transformation

For the transformation I did 6 samples:

  • 1 exp. Top10 (50uL), Positive control, negative control (25uL ea.)
  • 1 exp. Top10F (50uL), Positive control, negative control (25uL ea.)

Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb.

NOTE: I could of sworn i put SOC, though I found another thing of SOC in the heat plate which makes me question myself... has anyone else done transformations recently?
NOTE: Just in case I added 250uL more SOC 30 min into the incubation and created a duplicate set of plates. I will incubate a total of 1h30 and plate on two and pray it works =D

Re-run of gel from Tuesday, w/ undigesed... + gel purification of \XP KaiA

Click for legend


I gel purified the samples 2, 4, and 7; these are marked with a yellow sticker "Kai A \XP 8.11.06" in the Bundles of Joy" box in the cyanobacteria freezer.

Egel attempt of visualizing ligation products pre-transformation

Click for legend


Apparently ligation products don't visualize at all on gels (usually the ligation is transformed and plated ... etc., before it can be visualized; thus, having no products does not signify that the ligation did not work). =(

==Notes from peng== Today, we should miniprep in the morning the J04550 vector (I think that s what its called) which we innoculated yesterday, and then proceed cutting that with SP and ligating it into the KaiA \XP I prepared above - this will make our first workable construct if all goes well =D Also, the assays should be done, though prehaps we should make doing the above step a priority.

I may be in a bit later today, depending if I get out before daylight :D

To-do

  • KaiA + J04500 construct
    • Make frozen stocks of the J04500 transformants we grew up yesterday
    • Miniprep (10 mL total volume)
    • Digest J04500\S-P
    • Ligate J04500\S-P + KaiA\X-P
  • Digest assay and Western blot tutorial prep
    • Miniprep the R0010 + E0241 transformants we grew up yesterday (R0010 + E0241 is hereafter referred to as "GFP device") (8 mL total volume)
    • Perform digest assay on GFP device plasmids
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-10&oldid=59029"