Difference between revisions of "Team:U of Guelph/Experiments"

 
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<h1 class="descSub">Experiment Overview</h1>
 
<h1 class="descSub">Experiment Overview</h1>
 
<p class="descP">
 
<p class="descP">
−
Here we will outline in words the basic progression of each of our experiment in order of experiments conducted. If specific perimeters were used in each experiment note them.</p>
+
Our experimental goal this year was to clone <i>frc</i> and <i>oxc</i> into <i>E. coli</i> DH5&alpha; using pET-28a. To accomplish this goal we had <i>frc</i> and <i>oxc</i> synthesized by IDT and used PCR to add the correct restriction enzyme sites. Site directed mutagenesis was used to add the PstI cut site to pET-28a, then <i>frc</i> and <i>oxc</i> were inserted using standard restriction enzyme digests and ligation. pET-28a<i>frc</i> and pET-28a<i>oxc</i> were then transformed into DH5&alpha;.</p>  
 +
 
 
<h1 class="exEntery">Preparation of Competent Cells</h1>
 
<h1 class="exEntery">Preparation of Competent Cells</h1>
−
<p class="descP"> Here include information on both the DH5a and BL21 competent cell prep.</p>
+
<p class="descP"> This year we prepared two different types of competent cells, DH5&alpha; and BL21. We used a chemical competency method involving calcium chloride. As the final step in preparation all cells were frozen in either 50 &micro;l or 100 &micro;l volumes by submersion in liquid nitrogen. Competent cells were then stored in a -80&#176;C freezer for further use.</p>
 +
 
 
<h1 class="exEntery">pET-28a isolation</h1>
 
<h1 class="exEntery">pET-28a isolation</h1>
−
<p class="descP"> Here include information how we isolated and purrified pET-28a from DH5a/pET-28a</p>
+
<p class="descP"> pET-28a was purified from DH5&alpha;/pET-28a cells provided by Cezar Khursigara's Lab using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20&#176;C freezer for further use.</p>
 +
 
 
<h1 class="exEntery">Site Directed Mutagenesis of pET-28a</h1>
 
<h1 class="exEntery">Site Directed Mutagenesis of pET-28a</h1>
−
<p class="descP"> Here include information on how we mutated pET-28a to insert PstI RE site.</p>
+
<p class="descP">PCR was used to conduct site directed mutagenesis to add a PstI cut site to pET-28a. Two basepairs were changed at the wild type XhoI cut site to change the sequence from ctcgag to ctgcag. Primers were ordered from IDT.</p>
 +
 
 
<h1 class="exEntery">Confirmation of SDM Success</h1>
 
<h1 class="exEntery">Confirmation of SDM Success</h1>
−
<p class="descP"> Here include information on the steps we took to confirm that our SDM occurred correctly.</p>
+
<p class="descP">A double restriction enzyme digest was conducted to ensure that the PstI cut site had been added correctly. A single restriction enzyme digest and undigested plasmid were used as controls. All digestion products were run on an agarose gel and visualized using either Novel Juice (FroggaBio) or SyBr. Samples were frozen in a -20&#176;C freezer for further use.
−
<h1 class="exEntery">Isolation of pET-28a(PstI) from DH5a</h1>
+
</p>
−
<p class="descP"> Here include information on how we isolated and purrified pET-28a(PstI) from DH5a/pET-28a</p>
+
  
−
To be, or not to be: that is the question:
+
<h1 class="exEntery">Isolation of pET-28a(PstI) from DH5a</h1>
−
Whether ’tis nobler in the mind to suffer
+
<p class="descP"> pET-28a was purified from DH5&alpha;/pET-28a cells using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20&#176;C freezer for further use.</p>
−
The slings and arrows of outrageous fortune,
+
−
Or to take arms against a sea of troubles,
+
−
And by opposing end them? To die: to sleep;
+
−
No more; and by a sleep to say we end
+
−
The heart-ache and the thousand natural shocks
+
−
That flesh is heir to, ’tis a consummation
+
−
Devoutly to be wish’d. To die, to sleep;
+
−
To sleep: perchance to dream: ay, there’s the rub;
+
−
For in that sleep of death what dreams may come
+
−
When we have shuffled off this mortal coil,
+
−
Must give us pause: there’s the respect
+
−
That makes calamity of so long life;
+
−
For who would bear the whips and scorns of time,
+
−
The oppressor’s wrong, the proud man’s contumely,
+
−
The pangs of despised love, the law’s delay,
+
−
The insolence of office and the spurns
+
−
That patient merit of the unworthy takes,
+
−
When he himself might his quietus make
+
−
With a bare bodkin? who would fardels bear,
+
−
To grunt and sweat under a weary life,
+
−
But that the dread of something after death,
+
−
The undiscover’d country from whose bourn
+
−
No traveller returns, puzzles the will
+
−
And makes us rather bear those ills we have
+
−
Than fly to others that we know not of?
+
−
Thus conscience does make cowards of us all;
+
−
And thus the native hue of resolution
+
−
Is sicklied o’er with the pale cast of thought,
+
−
And enterprises of great pith and moment
+
−
With this regard their currents turn awry,
+
−
And lose the name of action.–Soft you now!
+
−
The fair Ophelia! Nymph, in thy orisons
+
−
Be all my sins remember’d.</p>
+
  
−
<h1 class="descSub">Protocols</h1>
+
<h1 class="exEntery">Addition of EcoRI and PstI to <i>frc</i> and <i>oxc</i></h1>
−
<p class="descP">
+
<p class="descP"> PCR was used to add EcoRI and PstI cut sites to the ends of <i>frc</i> and <i>oxc</i>. PCR primers and synthesized <i>frc</i> and <i>oxc</i> were all obtained from IDT.</p>
−
Here we will include a detailed list of all the basic protocols used in our experiments. Specific parameters for things such as PCRs will be noted above<br><br>
+
  
−
To be, or not to be: that is the question:
+
<h1 class="exEntery">Adding <i>frc</i> and <i>oxc</i> to pET-28a</h1>
−
Whether ’tis nobler in the mind to suffer
+
<p class="descP"> The genes <i>frc</i> and <i>oxc</i> were added individually to pET-28a using standard ligation techniques.
−
The slings and arrows of outrageous fortune,
+
</p>
−
Or to take arms against a sea of troubles,
+
<h1 class="exEntery">Transformation of pET-28a<i>frc</i> and pET-28a<i>oxc</i> into DH5&alpha;</h1>
−
And by opposing end them? To die: to sleep;
+
<p class="descP"> Using standard heat shock techniques pET-28a<i>frc</i> and pET-28a<i>oxc</i> were transformed into DH5&alpha;.
−
No more; and by a sleep to say we end
+
Transformed cells were incubated for 1 hour at 37&#176;C with rotation (using our homemade rotator) then plated on Kanamycin plates.</p>
−
The heart-ache and the thousand natural shocks
+
−
That flesh is heir to, ’tis a consummation
+
−
Devoutly to be wish’d. To die, to sleep;
+
−
To sleep: perchance to dream: ay, there’s the rub;
+
−
For in that sleep of death what dreams may come
+
−
When we have shuffled off this mortal coil,
+
−
Must give us pause: there’s the respect
+
−
That makes calamity of so long life;
+
−
For who would bear the whips and scorns of time,
+
−
The oppressor’s wrong, the proud man’s contumely,
+
−
The pangs of despised love, the law’s delay,
+
−
The insolence of office and the spurns
+
−
That patient merit of the unworthy takes,
+
−
When he himself might his quietus make
+
−
With a bare bodkin? who would fardels bear,
+
−
To grunt and sweat under a weary life,
+
−
But that the dread of something after death,
+
−
The undiscover’d country from whose bourn
+
−
No traveller returns, puzzles the will
+
−
And makes us rather bear those ills we have
+
−
Than fly to others that we know not of?
+
−
Thus conscience does make cowards of us all;
+
−
And thus the native hue of resolution
+
−
Is sicklied o’er with the pale cast of thought,
+
−
And enterprises of great pith and moment
+
−
With this regard their currents turn awry,
+
−
And lose the name of action.–Soft you now!
+
−
The fair Ophelia! Nymph, in thy orisons
+
−
Be all my sins remember’d.</p>
+
  
−
<h1 class="descSub">Refrences</h1>
+
<h1 class="exEntery">Confirmation of the Presence of <i>frc</i> and <i>oxc</i></h1>
−
<p class="descRef"> Rose, D. This is a test (2017). Sci. Awesome. 28-29 </p>
+
<p class="descRef"> Colony PCR was used to confirm the presence of <i>frc</i> and <i>oxc</i> from the colonies grown from the transformation. One of each type of colony was selected to make liquid culture, and then glycerol stocks from for long term storage of the transformed bacteria.</p>
  
 
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Latest revision as of 11:28, 31 October 2017

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Experiments

Experiment Overview

Our experimental goal this year was to clone frc and oxc into E. coli DH5α using pET-28a. To accomplish this goal we had frc and oxc synthesized by IDT and used PCR to add the correct restriction enzyme sites. Site directed mutagenesis was used to add the PstI cut site to pET-28a, then frc and oxc were inserted using standard restriction enzyme digests and ligation. pET-28afrc and pET-28aoxc were then transformed into DH5α.

Preparation of Competent Cells

This year we prepared two different types of competent cells, DH5α and BL21. We used a chemical competency method involving calcium chloride. As the final step in preparation all cells were frozen in either 50 µl or 100 µl volumes by submersion in liquid nitrogen. Competent cells were then stored in a -80°C freezer for further use.

pET-28a isolation

pET-28a was purified from DH5α/pET-28a cells provided by Cezar Khursigara's Lab using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20°C freezer for further use.

Site Directed Mutagenesis of pET-28a

PCR was used to conduct site directed mutagenesis to add a PstI cut site to pET-28a. Two basepairs were changed at the wild type XhoI cut site to change the sequence from ctcgag to ctgcag. Primers were ordered from IDT.

Confirmation of SDM Success

A double restriction enzyme digest was conducted to ensure that the PstI cut site had been added correctly. A single restriction enzyme digest and undigested plasmid were used as controls. All digestion products were run on an agarose gel and visualized using either Novel Juice (FroggaBio) or SyBr. Samples were frozen in a -20°C freezer for further use.

Isolation of pET-28a(PstI) from DH5a

pET-28a was purified from DH5α/pET-28a cells using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20°C freezer for further use.

Addition of EcoRI and PstI to frc and oxc

PCR was used to add EcoRI and PstI cut sites to the ends of frc and oxc. PCR primers and synthesized frc and oxc were all obtained from IDT.

Adding frc and oxc to pET-28a

The genes frc and oxc were added individually to pET-28a using standard ligation techniques.

Transformation of pET-28afrc and pET-28aoxc into DH5α

Using standard heat shock techniques pET-28afrc and pET-28aoxc were transformed into DH5α. Transformed cells were incubated for 1 hour at 37°C with rotation (using our homemade rotator) then plated on Kanamycin plates.

Confirmation of the Presence of frc and oxc

Colony PCR was used to confirm the presence of frc and oxc from the colonies grown from the transformation. One of each type of colony was selected to make liquid culture, and then glycerol stocks from for long term storage of the transformed bacteria.

University of Guelph iGEM 2017