Team:Alberta/Building Parts

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== Building Parts ==
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<div id="title">
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<p class="logo"><img src="https://static.igem.org/mediawiki/2010/c/cc/Alberta_Logo.png" width="50px" height="50px" padding="5px" align="left"></img></p>
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genomikon</div>
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<!-- NAVBAR -->
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<div id="navbar"><span class="llink2"><a href="https://2010.igem.org/Team:Alberta/JTest"> home</a> |  <a href="project"> project</a> | <a href="ethics"> ethics</a> | <a href="parts"> parts</a> | <a href="software"> software</a> | <a href="https://2010.igem.org/Team:Alberta/Notebook"> notebook</a> | <a href="team"> team</a></span>
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</div>
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=10-05-2010=
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<div id="Content">
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PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends.  
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<div id="vtab0">
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  <li onclick="showTab('vtab2')">Base Plasmids v.1</li>
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  <li onclick="showTab('vtab3')">Base Plasmids v.2</li>
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  <li onclick="showTab('vtab4')">Antibiotic Markers</li>
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  <li onclick="showTab('vtab5')">Abandoned Projects</li>
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</div>
   
   
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Recipe:
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<div id="tabContent">
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:1uL p1003 (approx. 1ng)
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:2.5uL prA_p1003+
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:2.5uL prB'_p1003-
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:5uL 10X PCR buffer
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:1uL 10uM dNTPs
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:2uL 50uM MgCl~2~
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:0.5uL Taq polymerase
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:35.5uL MilliQ H~2~O
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Program:
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<div id="blacktop">Building Parts</div>
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# 5 min-94^o^C
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# 45 sec-94^o^C
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<div id="vtab2" class="vtInfo"><span class="h2">Base Plasmids v.1</span>
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# 1 min-60^o^C
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# 1 min-72^o^C
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<p>Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI or BbsI cut sites.  If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky ends characteristic of an A or a B BioByte. </p>
 +
 
 +
<p><b>May 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-</p>
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<p><b>May 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-</p>
 +
<p><b>May 19, 2010</b> Digested pSB1C3 with Not1 </p>
 +
<p><b>May 19, 2010</b> Digested PCR products of p1003 (Kanamycin cassette) Not1 </p>
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<p><b>May 25, 2010</b> Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3 </p>
 +
</div>
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<div id="vtab3" class="vtInfo"><span class="h2">Testing Parts</span> 
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<p>Before we were able to test parts we created 2 base testing plasmids (vector 01 and vector 02).  Vector 01 is designed to test Open Reading Frame parts, or parts that code for proteins.  The part is flanked by a promoter and the start codon on one side and a stop codon and terminator on the other.  Vector 02 is designed to test linker parts, or parts that control the expression of the Open Reading Frame parts they are next two.  In Vector 02 the part is flanked by two distinct reporter genes, that by comparing the relative expression of the 2 reporter genes we can determine the behavior of the linking part.</p>
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</div>
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<div id="vtab4" class="vtInfo">
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<img src="https://static.igem.org/mediawiki/2010/b/b1/Alberta_Oscar.png" width="400px" height="533px" padding="0px" align="left"></img>
 +
<span class="h2">Oscar Cortes</span><br>
 +
<span class="h3">Specialized in Molecular Genetics (Graduate)</span>
 +
</div>
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<div id="footer"><span class="llink3">
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<a href="Site_Map">site map</a>|<a href="Sponsors">sponsors</a>|<a href="Contact">contact us</a>
 +
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</body>
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</html>
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== Building Parts ==
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__notoc__
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==10-05-2010==
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 +
<p>PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)</p>
 +
<p>Recipe:
 +
:1&mu;L p1003 (approx. 1ng)
 +
:2.5&mu;L prA_p1003+
 +
:2.5&mu;L prB'_p1003-
 +
:5&mu;L 10X PCR buffer
 +
:1&mu;L 10uM dNTPs
 +
:2&mu;L 50uM MgCl<sub>2</sub>
 +
:0.5&mu;L Taq polymerase
 +
:35.5&mu;L MilliQ H<sub>2</sub>O</p>
 +
</br>
 +
<p>Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-. </p>
 +
<p>Program:
 +
# 5 min-94<sup>o</sup>C
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# 45 sec-94<sup>o</sup>C
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# 1 min-60<sup>o</sup>C
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# 1 min-72<sup>o</sup>C
# Repeat 2 through 4 35 times
# Repeat 2 through 4 35 times
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# 5 min-72^o^C
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# 5 min-72<sup>o</sup>C</p>
 +
<!--Image of gel performed that day. 5&mu;L of each PCR reaction, 1&mu;L of 10X loading dye and 4&mu;L MilliQ water in a 1% agarose gel -->
-
<!--Image of gel performed that day. 5uL of each PCR reaction, 1uL of 10X loading dye and 4uL MilliQ water in a 1% agarose gel -->
+
==11-05-2010==
 +
<p>PCR purification of KanR A/B'-Bsa and KanR B/A'-Bsa created [[#10-05-2010|10-05-2010]] with Qiagen PCR cleanup kit.</p>
 +
<p>Determined concentrations by nanodrop.  KanA/B'-Bsa: 101.1ng/&mu;L KanB/A'-Bsa:89.6ng/&mu;L</p>
 +
==17-05-2010==
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<p>Innoculated 250mL overnight cultures with 10mL,4mL and 2mL of a starter culture of DH5&alpha;.  Left shaking at 18<sup>o</sup>C overnight. </p>
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=11-05-2010=
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==18-05-2010==
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<p>Prepared DH5&alpha; E.Coli competent cells using the Inoue Method. </p>
 +
<p>Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37<sup>o</sup>C on Chloramphenicol plates</p>
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PCR purification of Kanamycin Resistant fragments created [[#10-05-2010|10-05-2010]] with Qiagen PCR cleanup kit.
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==19-05-2010==
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Determined concentrations by nanodrop.  KanA/B': 101.1ng/uL KanB/A':89.6ng/uL
+
<p>From the transformation of DH5&alpha; cells with pSB1C3-J04450 performed on [[#18-05-2010|18-05-2010]], we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.</p>
-
=18-05-2010=
+
==20-05-2010==
 +
<p>Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5&alpha; cells with pSB1C3-J04450 from [[#19-05-2010|19-05-2010]].  Took a 1&mu;L sample of the Miniprep solutions and digested with NotI at 37<sup>o</sup>C for 1 hour. </p>
 +
<p>Digestion Recipe:
 +
:1&mu;L Miniprep (between 153.2 ng/&mu;l and 302.7ng/&mu;l determined by nanodrop)
 +
:1&mu;L NotI
 +
:1&mu;L 10X ReACT 3
 +
:7&mu;L MilliQ</p>
 +
<p>Ran Digestion on a 1% agarose gel to check that the plasmid obtained with what we expected. </p>
 +
<!-- Image of Gel-->
-
Prepared DH5&alpha; E.Coli competent cells using the Inoue Method.
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==25-05-2010==
-
Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37^o^C on Chloramphenicol plates
+
<p>Made 1.5mL LB liquid cultures of pSB1C3 from the plate streaked on [[#19-05-2010|19-05-2010]] and added chloramphenicol.</p>
-
=19-05-2010=
+
==26-05-2010==
 +
<p>Made 3 glycerol stocks of pSB1C3 from overnight made [[#25-05-2010|25-05-2010]]</p>
-
From the transformation of DH5&alpha; cells with pSB1C3-J04450 performed on [[#18-05-2010|18-05-2010]], we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.
+
==27-05-2010==
 +
</p>Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C.  Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C for 1 hour then took 15&mu;L to room temperature for 2 hours.  Transformed 100&mu;L of DH5&alpha; cells with 5&mu;L of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.</p>
 +
<p>Digestion Recipe:
 +
:1&mu;L Miniprep (302.7ng/&mu;l determined by nanodrop)
 +
:2&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)
 +
:1&mu;L NotI
 +
:1&mu;L 10X ReACT 3
 +
:5&mu;L MilliQ</p>
 +
<p>Ligation Recipe:
 +
:10&mu;L of Digest solution
 +
:1&mu;L T4 DNA ligase
 +
:6&mu;L 5X Buffer
 +
:13&mu;L MilliQ H<sub>2</sub>O</p>
 +
<p>Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5&alpha; cells.</p>
 +
<p>Performed PCR reactions to create parts with antibiotic resistance with negative controls.</p>
 +
<p>PCR Recipe:
 +
:3&mu;L 10X PCR Buffer
 +
:1&mu;L 10 uM dNTPs
 +
:2&mu;L 50 uM MgCl<sub>2</sub>
 +
:17.5&mu;L MilliQ H<sub>2</sub>O
 +
:0.5&mu;L Taq Polymerase
 +
:1&mu;L Template (psB4A5-J04450, psB4C5-J04450 or psB3T5-J04450)
 +
:2.5&mu;L Primer + (PrA psB4A5 ApR+, PrA psB4C5 ChR+ or PrA psB3T5 TR+)
 +
:2.5&mu;L Primer - (PrB psB4A5 ApR-, PrB psB4C5 ChR- or PrB psB3T5 TR-)</p>
 +
<p>PCR Program:
 +
# 5 min-94<sup>o</sup>C
 +
# 45 sec-94<sup>o</sup>C
 +
# 1 min-60<sup>o</sup>C
 +
# 1 min-72<sup>o</sup>C
 +
# Repeat 2 through 4 35 times
 +
# 5 min-72<sup>o</sup>C</p>
 +
<!--gel images of PCR Products (Alina's) and ligated and pre-digested samples (Jeremy's)-->
-
=20-05-2010=
+
==28-05-2010==
 +
<p>We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary</p>
-
Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5&alpha; cells with pSB1C3-J04450 from [[#19-05-2010|19-05-2010]].  Took a 1uL sample of the Miniprep solutions and digested with NotI at 37^o^C for 1 hour.  
+
==30-05-2010==
 +
<p>From the transformation of DH5&alpha; cells with pSB1C3-KanR performed on [[#28-05-2010|28-05-2010]], we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics overnight at 37<sup>o</sup>C. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.</p>
 +
 
 +
==31-05-2010==
 +
<p>9/12 of the pSB1C3-KanA/B' Liquid cultures [[#30-05-2010|30-05-2010]] were successful and only 1/12 of the pSB1C3-KanB/A' liquid cultures were successful.  The pSB4A5, pSB3T5 and pSB4C5 liquid cultures worked.  Miniprepped all the liquid cultures that worked. However, the streaks on the plates worked.</p>
 +
<p>Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps.  Digested with XbaI at 37<sup>o</sup>C for one hour and then with EcoRI at 37<sup>o</sup>C for one hour.  Ran a 1% agarose gel of the digests to determine the orientation of the KanR fragments. </p>
 +
<!--who did this digest!! we need an image-->
 +
<p>KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]], digested with BsaI at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>C for 30 minutes.  Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other.  Also tried to ligate KanA/B' fragments with KanB/A'.  Ligated with T4 DNA ligase overnight at 16<sup>o</sup>C.</p>
 +
 
 +
==01-06-2010==
 +
 
 +
KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]], digested with BsaI-HF at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>C for 30 minutes.  Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other.  Also tried to ligate KanA/B' fragments with KanB/A'.  Ligated with T4 DNA ligase for 3 hours at 21<sup>o</sup>C.
 +
 
 +
Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on [[#30-05-2010|30-05-2010]]
 +
 
 +
==02-06-2010==
 +
 
 +
Miniprepped liquid cultures from [[#01-06-2010|01-06-2010]]. Ran a 1% agarose gel of the ligations performed [[#01-06-2010|01-06-2010]].
 +
<!-- image of gel-->
 +
To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]] with the following recipe:
Digestion Recipe:
Digestion Recipe:
-
:1uL Miniprep (between 153.2 ng/ul and 302.7ng/ul determined by nanodrop)
+
:14&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)
-
:1uL NotI
+
:5&mu;L 1/10 dillution of 100X BSA
-
:1uL 10X ReACT 3
+
:5&mu;L 10X NEBuffer4
-
:7uL MilliQ
+
:1.5&mu:L BsaI-HF
 +
:24.5&mu;L MilliQ
 +
Digested at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 65<sup>o</sup>C for 20 minutes
 +
PCR purified the digests.
-
<!-- Image of Gel-->
+
==03-06-2010==
-
=27-05-2010=
+
Tried to ligate the KanA/B' fragments to itself.  Tried to ligate the KanB/A' fragments to itself.  Tried to ligate the KanA/B' fragments to the KanB/A' fragments.
-
Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37^o^C for 1 hour. Heat inactivated the NotI for 10 minutes at 65^o^C.  Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16^o^C for 1 hour then took 15uL to room temperature for 2 hours.  Transformed 100uL of DH5&alpha; cells with 5uL of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.
+
Ligation Recipe:
 +
:8&mu;L of digest from [[#02-06-2010|02-06-2010]] (either 8&mu;L of one of the fragments or 4&mu;L of each)
 +
:1&mu;L T4 DNA ligase
 +
:6&mu;L 5X Buffer
 +
:15&mu;L MilliQ H<sub>2</sub>O
 +
Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation. 
 +
<!--images-->
 +
 +
Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from [[#02-06-2010|02-06-2010]].
Digestion Recipe:
Digestion Recipe:
-
:1uL Miniprep (302.7ng/ul determined by nanodrop)
+
:14&mu;L plasmid (approx 300-400ng/&mu:L)
-
:2uL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/uL)
+
:5&mu;L 10X NEBuffer 4
-
:1uL NotI
+
:5&mu;L 1/10 dilution of 100X BSA
-
:1uL 10X ReACT 3  
+
:1.5&mu;L BsaI-HF
-
:5uL MilliQ
+
:24.5&mu;L MilliQ H<sub>2</sub>O
 +
 
 +
 
 +
Made a 1/100 dilution of AmpR and TetR PCR Products from [[#27-05-2010|27-05-2010]] and performed PCR reactions to produce antibiotic inserts to make parts.
 +
 
 +
PCR Recipe:
 +
:35.5&mu;L MilliQ H<sub>2</sub>O
 +
:1&mu;L 10 uM dNTPs
 +
:5&mu;L 10X PCR Buffer
 +
:2&mu;L 50 uM MgCl<sub>2</sub>
 +
:2.5&mu;L Primer + (ApR 1/10+ or TR 1/10+)
 +
:2.5&mu;L Primer - (ApR 1/10- or TR 1/10-)
 +
:1&mu;L Template (1/100 diluted AmpR or TetR)
 +
:0.5&mu;L Taq Polymerase
 +
 
 +
PCR Program:
 +
# 5 min-94<sup>o</sup>C
 +
# 45 sec-94<sup>o</sup>C
 +
# 1 min-60<sup>o</sup>C
 +
# 1 min-72<sup>o</sup>C
 +
# Repeat 2 through 4 35 times
 +
# 5 min-72<sup>o</sup>C
 +
 
 +
==04-06-2010==
 +
 
 +
Tried to test the limits of ligation reaction
 +
Ligation Recipe of plasmids cut on [[#03-06-2010|03-06-2010]]:
 +
:8&mu;L digestion mixture
 +
:6&mu;L 5X T4 ligase buffer
 +
:1&mu;L T4 ligase
 +
:15&mu;L MilliQ
 +
 
 +
Tried to further reaction of KanA/B' fragments to KanB/A' fragments.  To the existing Ligase reactions from [[#03-06-2010|03-06-2010]] added:
 +
:1&mu;L T4 ligase
 +
:6&mu;L 5X T4 ligase buffer
 +
:23&mu;L MilliQ
 +
 
 +
Tried to set limits of Kan fragments that would ligate. 
 +
:24&mu;L digestion from [[#02-06-2010|02-06-2010]] (either 24&mu;L of one of the fragments or 12&mu;L of each)
 +
:6&mu;L 5X T4 ligase buffer
 +
:1&mu;L T4 ligase
 +
 
 +
<!--images-->
 +
 
 +
==09-06-2010==
 +
 
 +
Restriction Digested AmpR and TetR inserts from [[#03-06-2010|03-06-2010]] to be ligated with psB1C3 vector later on.
 +
 
 +
Digestion Recipe for AmpR:
 +
:13.4&mu;L MilliQ H<sub>2</sub>O
 +
:0.60&mu:L AmpR (333.3 ng/&mu;L, determined by nanodrop)
 +
:2&mu;L 10X BSA
 +
:2&mu;L 10X Buffer 4
 +
:2&mu;L BsaI
 +
 
 +
Digestion Recipe fro TetR:
 +
:13.3&mu;L MilliQ H<sub>2</sub>O
 +
:0.7&mu:L TetR (571.3 ng/&mu;L, determined by nanodrop)
 +
:2&mu;L 10X BSA
 +
:2&mu;L 10X Buffer 4
 +
:2&mu;L BsaI
 +
 
 +
Both digestions were incubated at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 70<sup>o</sup>C for 20 minutes.
 +
 
 +
==10-06-2010==
 +
 
 +
Double Digested Kan/Chlor minipreps <!--Find date of Kan/Chlor production--> to determine orientation.
 +
 
 +
Digestion Recipe:
 +
:5&mu;L 10X Buffer 3
 +
:1&mu;L XbaI
 +
:1&mu;L PstI
 +
:5&mu;L Kan/Chlor fragments
 +
:33&mu;L MilliQ H<sub>2</sub>O
 +
:5&mu;L 10X BSA
 +
 
 +
Incubated at 37<sup>o</sup>C for 1hour, heat inactivated the enzyme at 80<sup>o</sup>C for 20 minutes.
 +
 
 +
<---gel image of 10.06.10 Karina1--->
 +
 +
 
 +
Restriction Digested A/B' psB1C3 vector.
 +
 
 +
Digestion Recipe:
 +
:10&mu;L MilliQ H<sub>2</sub>O
 +
:5&mu:L psB1C3
 +
:2&mu;L 10X BSA
 +
:2&mu;L 10X Buffer 4
 +
:1&mu;L BsaHF
 +
 
 +
Incubated at 37<sup>o</sup>C for 1hour, heat inactivated the enzyme at 80<sup>o</sup>C for 20 minutes.
 +
 
 +
Ligated digested A/B' psB1C3 with AmpR from [[#09-06-2010|09-06-2010]].
Ligation Recipe:
Ligation Recipe:
-
:10uL of Digest solution
+
:9&mu;L A/B' psB1C3 vector
-
:1uL T4 DNA ligase  
+
:5&mu;L AmpR insert
-
:6uL 5X Buffer
+
:6&mu;L 5X Ligase Buffer
-
:13uL MilliQ H~2~O
+
:1&mu;L Ligase
 +
:9&mu;L MilliQ H<sub>2</sub>O
 +
 
 +
Incubated at room temperature for 45 minutes. Transformed with DH5&alpha; cells using 15&mu;L of the ligated A/B' psB1C3 with AmpR.
 +
 
 +
==11-06-2010==
 +
 
 +
We got colonies of psB1C3 with AmpR from the DH5&alpha; transformations from [[#10-06-2010|10-06-2010]]. Hooray~ We made overnights.
 +
 
 +
==12-06-2010==
 +
 
 +
Miniprep of psB1C3 with AmpR was made from the overnights from [[#11-06-2010|11-06-2010]].
 +
 
 +
==16-06-2010==
 +
 
 +
psB1C3 with AmpR miniprep from [[#12-06-2010|12-06-2010]] ran undigested on a 1% agarose gel.
 +
 
 +
<!--gel image of Karina's 16.06.10-->
 +
 
 +
We digested psB1C3 with AmpR to see if the AmpR insert would be released from the psB1C3 vector.
 +
 
 +
Digestion Recipe:
 +
:14.1&mu;L MilliQ H<sub>2</sub>O
 +
:0.9&mu;L psB1C3 with AmpR (222.5 ng/&mu;L, determined by nanodrop)
 +
:2&mu;L 10X BSA
 +
:2&mu;L 10X Buffer 4
 +
:1&mu;L BsaI
 +
 
 +
Incubated at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 65<sup>o</sup>C for 30 minutes.
 +
 
 +
Also, we made more overnights of psB1C3 with AmpR.
 +
 
 +
==17-06-2010==
 +
 
 +
Digested psB1C3 with AmpR from [[#16-06-2010|16-06-2010]] was ran on a 1% agarose gel.
 +
 
 +
<!--17.06.10 Anh-->
 +
 
 +
==28-06-2010==
 +
<p>Transformed 2009 Cambridge color series parts:  Bba_K274100, BBa_K274200, BBa_K274002, BBa_K274003,  BBa_K274004, BBa_J23100.  Also transformed 2004 UTAustin BBa_M30109. Transformed using 5uL of DNA.</p>
 +
<p>Made starter culture of Dbl3 in LB at 11:03am.  Innoculated large overnight liquid culture as per specifications in Inoue method at 6:00pm</p>
 +
<p>Performed an enzyme efficiency experiment to determine which of BbsI, BfuAI, BsaI, or BsaI-HF works most efficiently.  </p>
 +
 
 +
==29-06-2010==
 +
 
 +
<p>Made a glycerol stock of PL5 from 1.5mL liquid overnight made [[#28-06-2010|28-06-2010]]. </p>
 +
<p> Took remaining transformation of Dbl3 cells with ccdB BfuA/B' and ccdB BfuB/A' from [[#28-06-2010|28-06-2010]] out of the fridge and plated 25, 50 and 100&mu;L of each on chloramphenicol LB plates</p>
 +
<p>From the chloramphenicol and chloramphenicol/Kanamycin plates streaked with ccdB BbsA/B', ccdB BbsB/A',ccdB BsaA/B' and  ccdB BsaB/A' on [[#28-06-2010|28-06-2010]], only ccdB BsaB/A' #6,8,10,11,12,14,15,18,  ccdB BbsA/B' #8,15 and 20 grew on only the chloramphenicol plate.  Made 5mL overnight liquid cultures with chloramphenicol of the some of the streaks that worked (ccdB BsaB/A' #6,8,10,11,12, ccdB BbsA/B' # 15, 20 and ccdB BsaB/A'#5 which didn't work but is a positive control).</p>
 +
<p>Miniprepped liquid culture grown [[#28-06-2010|28-06-2010]] of Kan BsaA/B' #7.  Concentration is 293.4ng/&mu;L.</p> 
 +
<p>All the transormations of Cambridge 1009 color series performed [[#28-06-2010|28-06-2010]] worked.  Made 5mL liquid cultures of each part.</p>
 +
<p>Made competent Dbl3 cells by the Inuoe method from liquid cultures grown overnight. (set up on [[#28-06-2010|28-06-2010]]). The OD of the culture was 0.775.</p>
 +
 
 +
==30-06-2010==
 +
 
 +
<p>Re Ran enzyme efficiency experiment with the same specifications as on [[#28-06-2010|28-06-2010]] but omitting BsaI-HF from the experiment because BsaI, BbsI and BfuAI appear to be the best of the enzymes.  Also we ran the experiment with 10 Units of each enzyme not 20 Units.</p>
 +
<p>Miniprepped liquid cultures of ccdB Bbs A/B' # 15 and 20 and of ccdB BsaB/A'#6, 8, 10, 11, 12 and 5(which did grow on both Kan/Chlor and Chlor plates, so it is a positive control for the incorrect plasmid) grown [[#29-06-2010|29-06-2010]].  The concentrations of the minipreps were between 200 and 400ng/mu;L and the curves did not show contamination.  Digested approximately 500ng of minipreps with EcoRI and PstI with the following recipe:
 +
:19.5&mu;L MilliQ H<sub>2</sub>O
 +
:2.5&mu:L Miniprep
 +
:3&mu;L 10X BSA
 +
:3&mu;L 10X NEBuffer 3
 +
:1&mu;L EcoRI
 +
:1&mu;L PstI
 +
</p>
 +
<p>made 5mL culture of Kan BbsA/B' #1 from [[#]]</p>
 +
<p>Ya! We got colonies of the ccdB BfuA/B' an ccdB BfuB/A' part plasmids that were plated [[#29-06-2010|29-06-2010]].</p>
 +
<p>Miniprep of liquid cultures made [[#29-06-2010|29-06-2010]] of transformations of Cambridge 2009 color series. Concentrations are as follows: J23100, 397.7 ng/&mu;L; K274100, 388.9ng/&mu;L; K274200, 556.5ng/&mu;L; K274004, 106.1ng/&mu;L; K130109, 82.5ng/&mu;L; K274003, 161.0ng/&mu;L.</p>
 +
 
 +
==01-07-2010==
 +
 
 +
Made 5mL liquid cultures with Choramphenicol with streaks from plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids from [[#25-06-2010|25-06-2010]].  Incubated at 37<sup>o</sup>C for 1 hour.
 +
Plated 25&mu;L of 1/100 and 1/1000 dilutions of liquid cultures onto Chloramphenicol LB plates.
 +
 
 +
Miniprepped liquid culture of Kan BbsA/B' #1 made [[#30-06-2010|30-06-2010]].
 +
 
 +
Ran gel of Enzyme efficiency experiment performed on [[#30-06-2010|30-06-2010]]. <!--Image w/ explanation-->
 +
 
 +
Ran gel of digests of Minipreps of ccdB part plasmids performed on [[#30-06-2010|30-06-2010]]to ensure they are as expected. <!-- Image w/ explanation-->
 +
 
 +
<!--AMANDA miniprep of ccdB BfuA/B', ccdB BfuB/A' part plasmids, digest with NotI?-->
 +
 
 +
==02-07-2010==
 +
 
 +
The colonies on the 1/100 and 1/1000 dilution plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids made on [[#01-07-2010|01-07-2010]], were spaced appropriately so as to be able to pick individual colonies.  We picked as many colonies as possible and streaked on both LB plates with Kanamycin and Chloramphenicol resistance and LB plates with only Choramphenicol resistance.  Again to determine if ccdB was infact inserted.
 +
 
 +
==Kan, Chlor, and Tet Parts==
 +
Strategy: to insert Kan into pSB1A3 after cutting both with NotI. Then, digest with BsaI to take Kan out and insert chlor and tet parts.
 +
 
 +
==30-06-2010==
 +
 
 +
=====Restriction Digest with NotI Protocol:=====
 +
 
 +
:Kan AB PCR product (82.3 ng/ul)    1ul
 +
:pSB1A3 plasmid    (30.5 ng/ul)    3ul
 +
:NotI enzyme                        1ul
 +
:10x REact3 buffer                  1ul
 +
:MilliQ H2O                        4ul
 +
:Total                            10ul
 +
:Incubated at 37C for 1 1/2 hours.
 +
 
 +
=====Ligation Protocol:=====
 +
 
 +
:Digested pSB1A3 and Kan AB fragment  8ul
 +
:T4 DNA ligase                       1ul
 +
:5X T4 DNA ligase buffer              6ul
 +
:MilliQ H20                          5ul
 +
:Total                              20ul
 +
:Incubated at room temperature for 1 hour.
 +
 
 +
=====Gel Electrophoresis:=====
 +
[[Image:Uofa_igem_29.06.10.alina2.jpg|thumb|none|300px|Lanes 2-4 are digests of pSB1A3 and kan AB fragment with NotI. Lanes 5-7 are ligations of the digested pSB1A3 and kan fragments. Note that no ligated DNA shows up on the gel due to a small volume of digestion (3ul) loaded.]]
 +
 
 +
=====Transformation:=====
 +
:Followed Inoue Protocol using 50ul DH5-alpha competent cells and 2ul of ligated pSB1A3 and kan AB part.
 +
:Plated 100ul, 100ul, and 400ul on Kan/Amp plates overnight at 37C.
 +
 
 +
:Results:
 +
::Plate 1 (100ul): TNTC white colonies and ~20 red colonies
 +
::Plate 2 (100ul): TNTC white colonies and ~3 red colonies
 +
::Plate 3 (400ul): TNTC white colonies and ~30 red colonies
 +
 
 +
==06-07-2010==
 +
 
 +
=====Overnights:=====
 +
:Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin.
 +
 
 +
:Results:
 +
::All seven tubes had growth and no growth in control tube.
 +
 
 +
 
 +
==07-07-2010==
 +
 
 +
=====Minipreps=====
 +
Followed Fermentas protocol to prepare 7 tubes.
 +
 
 +
=====Restriction Digest with XbaI and PstI Protocol to check for orientation:=====
 +
 
 +
:Kan AB in pSB1A3                  1ul
 +
:XbaI enzyme                        2ul
 +
:PstI enzyme                        1ul
 +
:NEBuffer 3                        4ul
 +
:MilliQ H2O                        3ul
 +
:BSA                                4ul
 +
:Total                            14ul
 +
:Incubated at 37C for 1 1/2 hours.
 +
 
 +
=====Gel Electrophoresis=====
 +
[[Image:Uofa_iGem_08.07,10alinakarina.jpg|thumb|none|300px|This is a shared gel. Lanes 2-8 are pSB1A3 plasmids with AB Kan inserts from different tubes, digested with XbaI and PstI. The plasmids from lanes 2 and 3 produced proper fragments. The rest of the tubes were discarded.]]
 +
 
 +
=====Restriction Digest with BsaI Protocol:=====
 +
 
 +
:Kan AB in pSB1A3 (247.4 ng/ul or 255.8 ng/ul)                1ul
 +
:chlor or tet AB fragment                                      1ul
 +
:BsaI enzyme                                                  0.5ul
 +
:NEBuffer 4                                                    3ul
 +
:MilliQ H2O                                                    5.5ul
 +
:Total                                                        11ul
 +
:Incubated at 50C for 1 hour.
 +
 
 +
=====Ligation Protocol:=====
 +
 
 +
:Digested pSB1A3 with Kan AB and chlor or tet          8ul
 +
:T4 DNA ligase                                        1ul
 +
:5X T4 DNA ligase buffer                              6ul
 +
:MilliQ H20                                            5ul
 +
:Total                                                20ul
 +
:Incubated at room temperature for 1 hour.
 +
 
 +
=====Gel Electrophoresis=====
 +
Insert gel here.
 +
 
 +
==08-07-2010==
 +
 
 +
=====Transformations=====
 +
:Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor.
 +
:Plated 200ul and 600ul on chlor/amp plates for chlor insert and on tet/amp plates for tet insert.
 +
 
 +
:Results:
 +
::chlor/amp plate 1 (200ul): ~300 colonies
 +
::chlor/amp plate 2 (600ul): TNTC colonies
 +
::tet/amp plate 1 (200ul): ~20 colonies
 +
::tet/amp plate 2 (600ul): ~30 colonies
 +
 
 +
==09-07-2010==
 +
 
 +
=====Overnights=====
 +
:5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp]
 +
 
 +
::Results
 +
:growth in all experimental tubes and no growth in control tubes
 +
 
 +
==10-07-2010==
 +
 
 +
=====Minipreps=====
 +
4 tubes of tet in pSB1A3 were prepared and 7 tubes of chlor in pSB1A3 were prepared.
 +
 
 +
==12-07-2010==
 +
 
 +
=====Restriction Digest with EcoRI Protocol to determine plasmid size:=====
 +
 
 +
:pSB1A3 with chlor or tet insert                              1ul                                   
 +
:Fast Digest EcoRI                                            1ul
 +
:10X Fast Digest Buffer                                        2ul
 +
:MilliQ H2O                                                    16ul
 +
:Total                                                        20ul
 +
:Incubated at 37C for 5 mins.
 +
 
 +
=====Gel Electrophoresis=====
 +
[[Image:Uofa_iGEM_12.07.10.alina.jpg|thumb|none|300px|Lanes 2-5 are pSB1A3 plasmid with tet AB insert. Lanes 6-13 are pSB1A3 plasmid with chlor AB insert. All plasmids were digested with EcoRI. All but the products of lanes 3,6,10,11,12 were correct. 1 out of the 4 tet in pSB1A3 tubes cut incorrectly and was discarded, 4 out of the 7 chlor in pSB1A3 tubes cut incorrectly and was discarded.]]
 +
 
 +
==14-07-2010==
 +
 
 +
=====Double Digest of chlor, kan and tet in PSB1A3 and amp is pSB1C3 with Xba and PstI protocol=====
-
=28-05-2010=
+
:plasmid with antibiotic part    1ul
 +
:10X NEBuffer 3                  1ul
 +
:MilliQ H20                      5ul
 +
:XbaI                            1ul
 +
:PstI                            1ul
 +
:10X BSA                        1ul
 +
:Total                          10ul
 +
:Incubated at 37C for 1 hour.
-
We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary
+
=====Gel Electrophoresis=====
 +
[[Image:Uofa igem 14.07.10.alina.jpg|thumb|none|300px|Digests of antibiotic AB fragments with XbaI and PstI. Lanes 2-3 are kanamycin AB, lanes 4-6 are chloramphenicol AB, lanes 7-9 are tetracycline AB, lanes 10-12 are ampicillin AB. All of the plasmids cut correctly except for one of the amp in pSB1C3, which was discarded]]
-
=30-05-2010=
+
==15-07-2010==
 +
:Antibiotic parts are now stored in the DNA box according to the BioBytes registry.
 +
:Glycerol stocks were made of overnights from chlor, tet, and kan in PSB1A3 plates.
-
From the transformation of DH5&alpha; cells with pSB1C3-KanR performed on [[#28-05-2010|28-05-2010]], we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics.
 
-
=31-05-2010=
+
{| style="color:#1b2c8a;background-color:#FFFF33;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
 +
!align="center"|[[Team:Alberta/Notebook|Notebook]]
 +
!align="center"|[[Team:Alberta/Building Parts|Building Parts]]
 +
!align="center"|[[Team:Alberta/Testing Parts|Testing Parts]]
 +
!align="center"|[[Team:Alberta/Assembly Method|Assembly Method]]
 +
!align="center"|[[Team:Alberta/Competent Cells|Competent Cells]]
 +
!align="center"|[[Team:Alberta/Plates|Plates]]
 +
!align="center"|[[Team:Alberta/Kit Manual|Kit Manual]]
 +
!align="center"|[[Team:Alberta/Software|Software]]
 +
|}

Latest revision as of 19:55, 19 August 2010

genomikon

  • Base Plasmids v.1
  • Base Plasmids v.2
  • Antibiotic Markers
  • Abandoned Projects
    • Building Parts
    Base Plasmids v.1

    Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI or BbsI cut sites. If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky ends characteristic of an A or a B BioByte.

    May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-

    May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-

    May 19, 2010 Digested pSB1C3 with Not1

    May 19, 2010 Digested PCR products of p1003 (Kanamycin cassette) Not1

    May 25, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3

    Testing Parts

    Before we were able to test parts we created 2 base testing plasmids (vector 01 and vector 02). Vector 01 is designed to test Open Reading Frame parts, or parts that code for proteins. The part is flanked by a promoter and the start codon on one side and a stop codon and terminator on the other. Vector 02 is designed to test linker parts, or parts that control the expression of the Open Reading Frame parts they are next two. In Vector 02 the part is flanked by two distinct reporter genes, that by comparing the relative expression of the 2 reporter genes we can determine the behavior of the linking part.

    Oscar Cortes
    Specialized in Molecular Genetics (Graduate)

    Building Parts

    10-05-2010

    PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)

    Recipe:

    1μL p1003 (approx. 1ng)
    2.5μL prA_p1003+
    2.5μL prB'_p1003-
    5μL 10X PCR buffer
    1μL 10uM dNTPs
    2μL 50uM MgCl2
    0.5μL Taq polymerase
    35.5μL MilliQ H2O

    </br>

    Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-.

    Program:

    1. 5 min-94oC
    2. 45 sec-94oC
    3. 1 min-60oC
    4. 1 min-72oC
    5. Repeat 2 through 4 35 times
    6. 5 min-72oC

    11-05-2010

    PCR purification of KanR A/B'-Bsa and KanR B/A'-Bsa created 10-05-2010 with Qiagen PCR cleanup kit.

    Determined concentrations by nanodrop. KanA/B'-Bsa: 101.1ng/μL KanB/A'-Bsa:89.6ng/μL

    17-05-2010

    Innoculated 250mL overnight cultures with 10mL,4mL and 2mL of a starter culture of DH5α. Left shaking at 18oC overnight.

    18-05-2010

    Prepared DH5α E.Coli competent cells using the Inoue Method.

    Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37oC on Chloramphenicol plates

    19-05-2010

    From the transformation of DH5α cells with pSB1C3-J04450 performed on 18-05-2010, we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.

    20-05-2010

    Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5α cells with pSB1C3-J04450 from 19-05-2010. Took a 1μL sample of the Miniprep solutions and digested with NotI at 37oC for 1 hour.

    Digestion Recipe:

    1μL Miniprep (between 153.2 ng/μl and 302.7ng/μl determined by nanodrop)
    1μL NotI
    1μL 10X ReACT 3
    7μL MilliQ

    Ran Digestion on a 1% agarose gel to check that the plasmid obtained with what we expected.

    25-05-2010

    Made 1.5mL LB liquid cultures of pSB1C3 from the plate streaked on 19-05-2010 and added chloramphenicol.

    26-05-2010

    Made 3 glycerol stocks of pSB1C3 from overnight made 25-05-2010

    27-05-2010

    </p>Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from 11-05-2010 and pSB1C3 from 20-05-2010 with NotI at 37oC for 1 hour. Heat inactivated the NotI for 10 minutes at 65oC. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16oC for 1 hour then took 15μL to room temperature for 2 hours. Transformed 100μL of DH5α cells with 5μL of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.</p>

    Digestion Recipe:

    1μL Miniprep (302.7ng/μl determined by nanodrop)
    2μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)
    1μL NotI
    1μL 10X ReACT 3
    5μL MilliQ

    Ligation Recipe:

    10μL of Digest solution
    1μL T4 DNA ligase
    6μL 5X Buffer
    13μL MilliQ H2O

    Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5α cells.

    Performed PCR reactions to create parts with antibiotic resistance with negative controls.

    PCR Recipe:

    3μL 10X PCR Buffer
    1μL 10 uM dNTPs
    2μL 50 uM MgCl2
    17.5μL MilliQ H2O
    0.5μL Taq Polymerase
    1μL Template (psB4A5-J04450, psB4C5-J04450 or psB3T5-J04450)
    2.5μL Primer + (PrA psB4A5 ApR+, PrA psB4C5 ChR+ or PrA psB3T5 TR+)
    2.5μL Primer - (PrB psB4A5 ApR-, PrB psB4C5 ChR- or PrB psB3T5 TR-)

    PCR Program:

    1. 5 min-94oC
    2. 45 sec-94oC
    3. 1 min-60oC
    4. 1 min-72oC
    5. Repeat 2 through 4 35 times
    6. 5 min-72oC

    28-05-2010

    We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary

    30-05-2010

    From the transformation of DH5α cells with pSB1C3-KanR performed on 28-05-2010, we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics overnight at 37oC. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.

    31-05-2010

    9/12 of the pSB1C3-KanA/B' Liquid cultures 30-05-2010 were successful and only 1/12 of the pSB1C3-KanB/A' liquid cultures were successful. The pSB4A5, pSB3T5 and pSB4C5 liquid cultures worked. Miniprepped all the liquid cultures that worked. However, the streaks on the plates worked.

    Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps. Digested with XbaI at 37oC for one hour and then with EcoRI at 37oC for one hour. Ran a 1% agarose gel of the digests to determine the orientation of the KanR fragments.

    KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI at 37oC for 1.5hours, heat inactivated at 65oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase overnight at 16oC.

    01-06-2010

    KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37oC for 1.5hours, heat inactivated at 65oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase for 3 hours at 21oC.

    Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on 30-05-2010

    02-06-2010

    Miniprepped liquid cultures from 01-06-2010. Ran a 1% agarose gel of the ligations performed 01-06-2010. To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on 11-05-2010 with the following recipe:

    Digestion Recipe:

    14μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)
    5μL 1/10 dillution of 100X BSA
    5μL 10X NEBuffer4
    1.5&mu:L BsaI-HF
    24.5μL MilliQ

    Digested at 50oC for 1hour, heat inactivated the enzyme at 65oC for 20 minutes PCR purified the digests.

    03-06-2010

    Tried to ligate the KanA/B' fragments to itself. Tried to ligate the KanB/A' fragments to itself. Tried to ligate the KanA/B' fragments to the KanB/A' fragments.

    Ligation Recipe:

    8μL of digest from 02-06-2010 (either 8μL of one of the fragments or 4μL of each)
    1μL T4 DNA ligase
    6μL 5X Buffer
    15μL MilliQ H2O

    Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.

    Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from 02-06-2010. Digestion Recipe:

    14μL plasmid (approx 300-400ng/&mu:L)
    5μL 10X NEBuffer 4
    5μL 1/10 dilution of 100X BSA
    1.5μL BsaI-HF
    24.5μL MilliQ H2O


    Made a 1/100 dilution of AmpR and TetR PCR Products from 27-05-2010 and performed PCR reactions to produce antibiotic inserts to make parts.

    PCR Recipe:

    35.5μL MilliQ H2O
    1μL 10 uM dNTPs
    5μL 10X PCR Buffer
    2μL 50 uM MgCl2
    2.5μL Primer + (ApR 1/10+ or TR 1/10+)
    2.5μL Primer - (ApR 1/10- or TR 1/10-)
    1μL Template (1/100 diluted AmpR or TetR)
    0.5μL Taq Polymerase

    PCR Program:

    1. 5 min-94oC
    2. 45 sec-94oC
    3. 1 min-60oC
    4. 1 min-72oC
    5. Repeat 2 through 4 35 times
    6. 5 min-72oC

    04-06-2010

    Tried to test the limits of ligation reaction Ligation Recipe of plasmids cut on 03-06-2010:

    8μL digestion mixture
    6μL 5X T4 ligase buffer
    1μL T4 ligase
    15μL MilliQ

    Tried to further reaction of KanA/B' fragments to KanB/A' fragments. To the existing Ligase reactions from 03-06-2010 added:

    1μL T4 ligase
    6μL 5X T4 ligase buffer
    23μL MilliQ

    Tried to set limits of Kan fragments that would ligate.

    24μL digestion from 02-06-2010 (either 24μL of one of the fragments or 12μL of each)
    6μL 5X T4 ligase buffer
    1μL T4 ligase


    09-06-2010

    Restriction Digested AmpR and TetR inserts from 03-06-2010 to be ligated with psB1C3 vector later on.

    Digestion Recipe for AmpR:

    13.4μL MilliQ H2O
    0.60&mu:L AmpR (333.3 ng/μL, determined by nanodrop)
    2μL 10X BSA
    2μL 10X Buffer 4
    2μL BsaI

    Digestion Recipe fro TetR:

    13.3μL MilliQ H2O
    0.7&mu:L TetR (571.3 ng/μL, determined by nanodrop)
    2μL 10X BSA
    2μL 10X Buffer 4
    2μL BsaI

    Both digestions were incubated at 50oC for 1hour, heat inactivated the enzyme at 70oC for 20 minutes.

    10-06-2010

    Double Digested Kan/Chlor minipreps to determine orientation.

    Digestion Recipe:

    5μL 10X Buffer 3
    1μL XbaI
    1μL PstI
    5μL Kan/Chlor fragments
    33μL MilliQ H2O
    5μL 10X BSA

    Incubated at 37oC for 1hour, heat inactivated the enzyme at 80oC for 20 minutes.

    <---gel image of 10.06.10 Karina1--->


    Restriction Digested A/B' psB1C3 vector.

    Digestion Recipe:

    10μL MilliQ H2O
    5&mu:L psB1C3
    2μL 10X BSA
    2μL 10X Buffer 4
    1μL BsaHF

    Incubated at 37oC for 1hour, heat inactivated the enzyme at 80oC for 20 minutes.

    Ligated digested A/B' psB1C3 with AmpR from 09-06-2010.

    Ligation Recipe:

    9μL A/B' psB1C3 vector
    5μL AmpR insert
    6μL 5X Ligase Buffer
    1μL Ligase
    9μL MilliQ H2O

    Incubated at room temperature for 45 minutes. Transformed with DH5α cells using 15μL of the ligated A/B' psB1C3 with AmpR.

    11-06-2010

    We got colonies of psB1C3 with AmpR from the DH5α transformations from 10-06-2010. Hooray~ We made overnights.

    12-06-2010

    Miniprep of psB1C3 with AmpR was made from the overnights from 11-06-2010.

    16-06-2010

    psB1C3 with AmpR miniprep from 12-06-2010 ran undigested on a 1% agarose gel.


    We digested psB1C3 with AmpR to see if the AmpR insert would be released from the psB1C3 vector.

    Digestion Recipe:

    14.1μL MilliQ H2O
    0.9μL psB1C3 with AmpR (222.5 ng/μL, determined by nanodrop)
    2μL 10X BSA
    2μL 10X Buffer 4
    1μL BsaI

    Incubated at 50oC for 1hour, heat inactivated the enzyme at 65oC for 30 minutes.

    Also, we made more overnights of psB1C3 with AmpR.

    17-06-2010

    Digested psB1C3 with AmpR from 16-06-2010 was ran on a 1% agarose gel.


    28-06-2010

    Transformed 2009 Cambridge color series parts: Bba_K274100, BBa_K274200, BBa_K274002, BBa_K274003, BBa_K274004, BBa_J23100. Also transformed 2004 UTAustin BBa_M30109. Transformed using 5uL of DNA.

    Made starter culture of Dbl3 in LB at 11:03am. Innoculated large overnight liquid culture as per specifications in Inoue method at 6:00pm

    Performed an enzyme efficiency experiment to determine which of BbsI, BfuAI, BsaI, or BsaI-HF works most efficiently.

    29-06-2010

    Made a glycerol stock of PL5 from 1.5mL liquid overnight made 28-06-2010.

    Took remaining transformation of Dbl3 cells with ccdB BfuA/B' and ccdB BfuB/A' from 28-06-2010 out of the fridge and plated 25, 50 and 100μL of each on chloramphenicol LB plates

    From the chloramphenicol and chloramphenicol/Kanamycin plates streaked with ccdB BbsA/B', ccdB BbsB/A',ccdB BsaA/B' and ccdB BsaB/A' on 28-06-2010, only ccdB BsaB/A' #6,8,10,11,12,14,15,18, ccdB BbsA/B' #8,15 and 20 grew on only the chloramphenicol plate. Made 5mL overnight liquid cultures with chloramphenicol of the some of the streaks that worked (ccdB BsaB/A' #6,8,10,11,12, ccdB BbsA/B' # 15, 20 and ccdB BsaB/A'#5 which didn't work but is a positive control).

    Miniprepped liquid culture grown 28-06-2010 of Kan BsaA/B' #7. Concentration is 293.4ng/μL.

    All the transormations of Cambridge 1009 color series performed 28-06-2010 worked. Made 5mL liquid cultures of each part.

    Made competent Dbl3 cells by the Inuoe method from liquid cultures grown overnight. (set up on 28-06-2010). The OD of the culture was 0.775.

    30-06-2010

    Re Ran enzyme efficiency experiment with the same specifications as on 28-06-2010 but omitting BsaI-HF from the experiment because BsaI, BbsI and BfuAI appear to be the best of the enzymes. Also we ran the experiment with 10 Units of each enzyme not 20 Units.

    Miniprepped liquid cultures of ccdB Bbs A/B' # 15 and 20 and of ccdB BsaB/A'#6, 8, 10, 11, 12 and 5(which did grow on both Kan/Chlor and Chlor plates, so it is a positive control for the incorrect plasmid) grown 29-06-2010. The concentrations of the minipreps were between 200 and 400ng/mu;L and the curves did not show contamination. Digested approximately 500ng of minipreps with EcoRI and PstI with the following recipe:

    19.5μL MilliQ H2O
    2.5&mu:L Miniprep
    3μL 10X BSA
    3μL 10X NEBuffer 3
    1μL EcoRI
    1μL PstI

    made 5mL culture of Kan BbsA/B' #1 from #

    Ya! We got colonies of the ccdB BfuA/B' an ccdB BfuB/A' part plasmids that were plated 29-06-2010.

    Miniprep of liquid cultures made 29-06-2010 of transformations of Cambridge 2009 color series. Concentrations are as follows: J23100, 397.7 ng/μL; K274100, 388.9ng/μL; K274200, 556.5ng/μL; K274004, 106.1ng/μL; K130109, 82.5ng/μL; K274003, 161.0ng/μL.

    01-07-2010

    Made 5mL liquid cultures with Choramphenicol with streaks from plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids from 25-06-2010. Incubated at 37oC for 1 hour. Plated 25μL of 1/100 and 1/1000 dilutions of liquid cultures onto Chloramphenicol LB plates.

    Miniprepped liquid culture of Kan BbsA/B' #1 made 30-06-2010.

    Ran gel of Enzyme efficiency experiment performed on 30-06-2010.

    Ran gel of digests of Minipreps of ccdB part plasmids performed on 30-06-2010to ensure they are as expected.


    02-07-2010

    The colonies on the 1/100 and 1/1000 dilution plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids made on 01-07-2010, were spaced appropriately so as to be able to pick individual colonies. We picked as many colonies as possible and streaked on both LB plates with Kanamycin and Chloramphenicol resistance and LB plates with only Choramphenicol resistance. Again to determine if ccdB was infact inserted.

    Kan, Chlor, and Tet Parts

    Strategy: to insert Kan into pSB1A3 after cutting both with NotI. Then, digest with BsaI to take Kan out and insert chlor and tet parts.

    30-06-2010

    Restriction Digest with NotI Protocol:
    Kan AB PCR product (82.3 ng/ul) 1ul
    pSB1A3 plasmid (30.5 ng/ul) 3ul
    NotI enzyme 1ul
    10x REact3 buffer 1ul
    MilliQ H2O 4ul
    Total 10ul
    Incubated at 37C for 1 1/2 hours.
    Ligation Protocol:
    Digested pSB1A3 and Kan AB fragment 8ul
    T4 DNA ligase 1ul
    5X T4 DNA ligase buffer 6ul
    MilliQ H20 5ul
    Total 20ul
    Incubated at room temperature for 1 hour.
    Gel Electrophoresis:
    Lanes 2-4 are digests of pSB1A3 and kan AB fragment with NotI. Lanes 5-7 are ligations of the digested pSB1A3 and kan fragments. Note that no ligated DNA shows up on the gel due to a small volume of digestion (3ul) loaded.
    Transformation:
    Followed Inoue Protocol using 50ul DH5-alpha competent cells and 2ul of ligated pSB1A3 and kan AB part.
    Plated 100ul, 100ul, and 400ul on Kan/Amp plates overnight at 37C.
    Results:
    Plate 1 (100ul): TNTC white colonies and ~20 red colonies
    Plate 2 (100ul): TNTC white colonies and ~3 red colonies
    Plate 3 (400ul): TNTC white colonies and ~30 red colonies

    06-07-2010

    Overnights:
    Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin.
    Results:
    All seven tubes had growth and no growth in control tube.


    07-07-2010

    Minipreps

    Followed Fermentas protocol to prepare 7 tubes.

    Restriction Digest with XbaI and PstI Protocol to check for orientation:
    Kan AB in pSB1A3 1ul
    XbaI enzyme 2ul
    PstI enzyme 1ul
    NEBuffer 3 4ul
    MilliQ H2O 3ul
    BSA 4ul
    Total 14ul
    Incubated at 37C for 1 1/2 hours.
    Gel Electrophoresis
    This is a shared gel. Lanes 2-8 are pSB1A3 plasmids with AB Kan inserts from different tubes, digested with XbaI and PstI. The plasmids from lanes 2 and 3 produced proper fragments. The rest of the tubes were discarded.
    Restriction Digest with BsaI Protocol:
    Kan AB in pSB1A3 (247.4 ng/ul or 255.8 ng/ul) 1ul
    chlor or tet AB fragment 1ul
    BsaI enzyme 0.5ul
    NEBuffer 4 3ul
    MilliQ H2O 5.5ul
    Total 11ul
    Incubated at 50C for 1 hour.
    Ligation Protocol:
    Digested pSB1A3 with Kan AB and chlor or tet 8ul
    T4 DNA ligase 1ul
    5X T4 DNA ligase buffer 6ul
    MilliQ H20 5ul
    Total 20ul
    Incubated at room temperature for 1 hour.
    Gel Electrophoresis

    Insert gel here.

    08-07-2010

    Transformations
    Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor.
    Plated 200ul and 600ul on chlor/amp plates for chlor insert and on tet/amp plates for tet insert.
    Results:
    chlor/amp plate 1 (200ul): ~300 colonies
    chlor/amp plate 2 (600ul): TNTC colonies
    tet/amp plate 1 (200ul): ~20 colonies
    tet/amp plate 2 (600ul): ~30 colonies

    09-07-2010

    Overnights
    5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp]
    Results
    growth in all experimental tubes and no growth in control tubes

    10-07-2010

    Minipreps

    4 tubes of tet in pSB1A3 were prepared and 7 tubes of chlor in pSB1A3 were prepared.

    12-07-2010

    Restriction Digest with EcoRI Protocol to determine plasmid size:
    pSB1A3 with chlor or tet insert 1ul
    Fast Digest EcoRI 1ul
    10X Fast Digest Buffer 2ul
    MilliQ H2O 16ul
    Total 20ul
    Incubated at 37C for 5 mins.
    Gel Electrophoresis
    Lanes 2-5 are pSB1A3 plasmid with tet AB insert. Lanes 6-13 are pSB1A3 plasmid with chlor AB insert. All plasmids were digested with EcoRI. All but the products of lanes 3,6,10,11,12 were correct. 1 out of the 4 tet in pSB1A3 tubes cut incorrectly and was discarded, 4 out of the 7 chlor in pSB1A3 tubes cut incorrectly and was discarded.

    14-07-2010

    Double Digest of chlor, kan and tet in PSB1A3 and amp is pSB1C3 with Xba and PstI protocol
    plasmid with antibiotic part 1ul
    10X NEBuffer 3 1ul
    MilliQ H20 5ul
    XbaI 1ul
    PstI 1ul
    10X BSA 1ul
    Total 10ul
    Incubated at 37C for 1 hour.
    Gel Electrophoresis
    Digests of antibiotic AB fragments with XbaI and PstI. Lanes 2-3 are kanamycin AB, lanes 4-6 are chloramphenicol AB, lanes 7-9 are tetracycline AB, lanes 10-12 are ampicillin AB. All of the plasmids cut correctly except for one of the amp in pSB1C3, which was discarded

    15-07-2010

    Antibiotic parts are now stored in the DNA box according to the BioBytes registry.
    Glycerol stocks were made of overnights from chlor, tet, and kan in PSB1A3 plates.


    Notebook Building Parts Testing Parts Assembly Method Competent Cells Plates Kit Manual Software
    Retrieved from "http://2010.igem.org/Team:Alberta/Building_Parts"