Team:Alberta/Notebook July

From 2010.igem.org

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Latest revision as of 22:55, 10 August 2010

genomikon

July 2010

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Notebook
Building Parts
Testing Parts
Assembly Method
Plates
Competent Cells
Software

iGEM 2010 Notebook

The lab notebook chronicles our journey in the creation of the Genomikon kit. Many paths were woven together in space and time to reach this finished masterpiece. To help you navigate through these trials with us we have laid out our notebook in a layered fashion. This page gives a sketch of each project and how it interacts with each other. Then follow the links to a projects page for time line of the major landmarks and accomplishments. If you require more details on the project the links within that page will take you to our day-by-day work log.

Building Parts

The Building Parts project was responsible to first build a plasmid (plasmid 01)that contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix. After plasmid 01 existed we inserted the CcdB gene (the "death" gene) between our prefix and suffix removing the gene for Kanamycin resistance (plasmid 02). Plasmid 02 is fantastic base plasmid from which we are able to amplify any part at all because it provides a positive selection marker when transformed into DH5α. At this point we were able to make parts en masse to put in our kit. After obtaining a particular part in a plasmid we PCRed the part and digested it ready to use in Assembly or to Test the plasmid.

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.

Assembly Method

Insert description here.

Plates

Insert description here.

Competent Cells

Insert description here.

Software

Insert description here.

July 1, 2010

Building Parts

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 37^oC 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.

<--Image w/ explanation-->

Ran gel of digests of Minipreps of ccdB part plasmids performed on 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?-->

July 2, 2010

Building Parts

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.

July 6, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin from plates made on 30-06-2010. Results: All seven tubes had growth and no growth in control tube.

Experiment continues on 07-07-2010.

July 7, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

We made minipreps from the overnights from 06-07-2010. 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.

Experiment continues on 08-07-2010.

July 8, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor made on 07-07-2010. 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

Experiment continues on 09-07-2010.

July 9, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Made overnights with 5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp] using transformation plates from 08-07-2010. Results were growth in all experimental tubes and no growth in control tubes.

Experiment continues on 10-07-2010.

July 10, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

We prepared minipreps of tet in pSB1A3 (4 tubes) and chlor in pSB1A3 (7 tubes) from overnights 09-07-2010.

Experiment continues on 12-07-2010.

July 12, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Restriction Digest with EcoRI Protocol to determine plasmid size:

10-07-2010

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.

Experiment continues on 14-07-2010.

July 14, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

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.

Experiment continues on 15-07-2010.

July 15, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Antibiotic parts, made on 14-07-2010, 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.

July 19, 2010

Building Parts

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Digest of pSB1A3 and premade Bfu AB kan fragments with NotI protocol:

pSB1A3 (30.5ng/ul) 3ul

10X REact 3 Buffer 1ul

MilliQ H20 10ul

Bfu Kan AB (38.1ng/ul) 3ul

NotI 1ul

10X BSA 2ul

Total 20ul

Incubated at 37C for 1 hour.

Digest of pSB1A3 and premade Bfu BA kan fragments with NotI protocol:

pSB1A3 (30.5ng/ul) 3ul

10X REact 3 Buffer 1ul

MilliQ H20 10ul

Bfu Kan BA 2ul

NotI 2ul

10X BSA 2ul

Total 20ul

Incubated at 37C for 1 hour.

Ligation of pSB1A3 and premade Bfu AB/BA kan fragments:

Digests from above 10ul

5X Ligase Buffer 4ul

MilliQ H20 5ul

T4 DNA ligase 1ul

Total 20ul

Incubated at room temperature for 1 hour.

July 20, 2010

Building Parts

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with kan AB or BA. Plated on kan/amp plates.

July 21, 2010

Building Parts

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Transformation results:

kan AB 1: 3 white colonies

kan AB 2: 50 white, 1 red colonies

kan AB 3: 45 white, 2 red colonies

kan BA 1: 25 white colonies

kan BA 2: 17 white, 1 red colonies

kan BA 3: 25 white, 1 red colonies

Set up overnights with 5 ul kan and 2.5 ul amp per 5ml LB

July 22, 2010

Building Parts

Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3

Miniprepped overnight tubes (growth in all).

Digest of pSB1A3 with Bfu kan AB/BA inserts with XbaI and PstI enzymes protocol:

plasmid DNA ul

10X NEBuffer 3 2ul

MilliQ H20 12ul

XbaI 1ul

Pst I 1ul

10X BSA 2ul

Total 20ul

Incubated for 1 hour at 37C.

Insert gel here

July 23, 2010

Building Parts

Constructing Bfu AB/BA chlor, amp and tet parts

Digest of Bfu Kan AB/BA in pSB1A3 and pSB1C3 with BfuAI protocol:

psb1A3 with Bfu Kan AB/BA insert OR psb1C3 with Bfu Kan AB/BA insert 5ul

BfuAI 1ul

NEBuffer 3 10ul

MilliQ H20 84ul

Total 100ul

Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.

Insert gel here

Digest of Bsa AB chlor, tet and amp in PSB1C3 with BsaI protocol:

psb1C3 with Bsa AB chlor, tet and amp 5ul

BsaI 1ul

NEBuffer 4 10ul

MilliQ H20 84ul

Total 100ul

Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.

Insert gel here

Ligations of Bfu amp AB/BA into pSB1C3 and Bfu chlor and tet AB/BA into pSB1A3 protocol:

pSB1A3 or pSB1C3 10ul

antibiotic insert 10ul

5x ligase buffer 8ul

T4 DNA ligase 1ul

Total 40ul

Incubate at room temperature for one hour.

July 26, 2010

Building Parts

Constructing Bfu AB/BA chlor, amp and tet parts

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 or pSB1C3 ligated with AB and BA antibiotics. Plated on various antibiotic plates.

July 28, 2010

Building Parts

Constructing Bfu AB/BA chlor, amp and tet parts

Overnights Results

Growth: Bfu AB amp1,2,3; Bfu AB chlor1,2,3; Bfu BA chlor1,2

No growth: Bfu AB chlor2, Bfu BA chlor3

Tubes with growth were miniprepped.

@@ Line 235: / Line 235: @@
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+      <li class="day"><a title="" onclick="return toggleMe('div22')" return false;" href="#" class="a1">22</a></li>
-      <li class="day"><a title="" onclick="return toggleMe('div23')" return false;" href="#" class="na">23</a></li>
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-      <li class="day"><a title="" onclick="return toggleMe('div26')" return false;" href="#" class="na">26</a></li>
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-      <li class="day"><a title="" onclick="return toggleMe('div27')" return false;" href="#" class="na">27</a></li>
+      <li class="day"><a title="" onclick="return toggleMe('div27')" return false;" href="#" class="a2">27</a></li>
-      <li class="day"><a title="" onclick="return toggleMe('div28')" return false;" href="#" class="na">28</a></li>
+      <li class="day"><a title="" onclick="return toggleMe('div28')" return false;" href="#" class="a1">28</a></li>
       <li class="day"><a title="" onclick="return toggleMe('div29')" return false;" href="#" class="na">29</a></li>
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@@ Line 439: / Line 441: @@
 <div class="tabContent" id="Notebook">
-<span class="h2">iGEM 2010 Notebook</span>
+<span class="h3">iGEM 2010 Notebook</span>
 <p>The lab notebook chronicles our journey in the creation of the Genomikon kit.  Many paths were woven together in space and time to reach this finished masterpiece.  To help you navigate through these trials with us we have laid out our notebook in a layered fashion.  This page gives a sketch of each project and how it interacts with each other.  Then follow the links to a projects page for time line of the major landmarks and accomplishments.  If you require more details on the project the links within that page will take you to our day-by-day work log.
@@ Line 446: / Line 448: @@
 <div class="tabContent" id="Building">
-<span class="h2">Building Parts</span>
+<span class="h3">Building Parts</span>
 <p>The Building Parts project was responsible to first build a plasmid (plasmid 01)that contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix.  After plasmid 01 existed we inserted the CcdB gene (the "death" gene) between our prefix and suffix removing the gene for Kanamycin resistance (plasmid 02).  Plasmid 02 is fantastic base plasmid from which we are able to amplify any part at all because it provides a positive selection marker when transformed into DH5&alpha;. At this point we were able to make parts en masse to put in our kit. After obtaining a particular part in a plasmid we PCRed the part and digested it ready to use in Assembly or to Test the plasmid.</p>
@@ Line 452: / Line 454: @@
 <div class="tabContent" id="Testing">
-<span class="h2">Testing Parts</span>
+<span class="h3">Testing Parts</span>
 <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>
@@ Line 458: / Line 460: @@
 <div class="tabContent" id="Assembly">
-<span class="h2">Assembly Method</span>
+<span class="h3">Assembly Method</span>
 <p>Insert description here.</p>
@@ Line 464: / Line 466: @@
 <div class="tabContent" id="Plates">
-<span class="h2">Plates</span>
+<span class="h3">Plates</span>
 <p>Insert description here.</p>
@@ Line 470: / Line 472: @@
 <div class="tabContent" id="Cells">
-<span class="h2">Competent Cells</span>
+<span class="h3">Competent Cells</span>
 <p>Insert description here.</p>
@@ Line 476: / Line 478: @@
 <div class="tabContent" id="Software">
-<span class="h2">Software</span>
+<span class="h3">Software</span>
 <p>Insert description here.</p>
@@ Line 507: / Line 509: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin from plates made on <a style="color: #fff200" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_June#30" onclick="" return false;">30-06-2010</a>. Results: All seven tubes had growth and no growth in control tube.</p>
@@ Line 517: / Line 519: @@
 <p>July 7, 2010</p><br>
 <span class="h2">Building Parts</span>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>We made minipreps from the overnights from <a style="color: #fff200" href="" onclick="return toggleMe('div6')" return false;">06-07-2010</a>. Followed Fermentas protocol to prepare 7 tubes. Restriction Digest with XbaI and PstI Protocol to check for orientation:
 <ul>Kan AB in pSB1A3 1ul</ul>
@@ Line 554: / Line 557: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor made on <a style="color: #fff200" href="" onclick="return toggleMe('div7')" return false;">07-07-2010</a>. Plated 200ul and 600ul on chlor/amp plates for chlor insert and on tet/amp plates for tet insert. Results:       <ul>chlor/amp plate 1 (200ul): ~300 colonies </ul>
@@ Line 569: / Line 572: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>Made overnights with 5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp] using transformation plates from <a style="color: #fff200" href="" onclick="return toggleMe('div8')" return false;">08-07-2010</a>. Results were growth in all experimental tubes and no growth in control tubes. </p>
@@ Line 580: / Line 583: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p> We prepared minipreps of tet in pSB1A3 (4 tubes) and chlor in pSB1A3 (7 tubes) from overnights <a style="color: #fff200" href="" onclick="return toggleMe('div9')" return false;">09-07-2010</a>.</p>
@@ Line 591: / Line 594: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>Restriction Digest with EcoRI Protocol to determine plasmid size:
@@ Line 611: / Line 614: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p>Double Digest of chlor, kan and tet in PSB1A3 and amp is pSB1C3 with Xba and PstI protocol:
@@ Line 633: / Line 636: @@
 <span class="h2">Building Parts</span>
-<p><span class="h1">Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone</span></p>
+<p><span class="h1">Constructing Bsa AB Kan, Chlor and Tet Parts</span></p>
 <p> Antibiotic parts, made on <a style="color: #fff200" href="" onclick="return toggleMe('div14')" return false;">14-07-2010</a>, are now stored in the DNA box according to the BioBytes registry.</p>
@@ Line 639: / Line 642: @@
 </div>
+<div align="left" id='div19' class="hideme">
+<p>July 19, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3</span></p>
+<p>Digest of pSB1A3 and premade Bfu AB kan fragments with NotI protocol:
+<ul>pSB1A3 (30.5ng/ul) 3ul </ul>
+<ul>10X REact 3 Buffer 1ul </ul>
+<ul>MilliQ H20 10ul </ul>
+<ul> Bfu Kan AB (38.1ng/ul) 3ul </ul>
+<ul>NotI 1ul </ul>
+<ul>10X BSA 2ul </ul>
+<ul>Total 20ul </ul></p>
+<p>Incubated at 37C for 1 hour.</p>
+<p>Digest of pSB1A3 and premade Bfu BA kan fragments with NotI protocol:
+<ul>pSB1A3 (30.5ng/ul) 3ul </ul>
+<ul>10X REact 3 Buffer 1ul </ul>
+<ul>MilliQ H20 10ul </ul>
+<ul> Bfu Kan BA 2ul </ul>
+<ul>NotI 2ul </ul>
+<ul>10X BSA 2ul </ul>
+<ul>Total 20ul </ul></p>
+<p>Incubated at 37C for 1 hour.</p>
+<p>Ligation of pSB1A3 and premade Bfu AB/BA kan fragments:
+<ul>Digests from above 10ul </ul>
+<ul>5X Ligase Buffer 4ul </ul>
+<ul>MilliQ H20 5ul </ul>
+<ul>T4 DNA ligase 1ul </ul>
+<ul>Total 20ul </ul></p>
+Incubated at room temperature for 1 hour.
+</div>
+<div align="left" id='div20' class="hideme">
+<p>July 20, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3</span></p>
+<p>Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with kan AB or BA. Plated on kan/amp plates.
+</div>
+<div align="left" id='div21' class="hideme">
+<p>July 21, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3</span></p>
+Transformation results:
+<ul>kan AB 1: 3 white colonies </ul>
+<ul>kan AB 2: 50 white, 1 red colonies </ul>
+<ul>kan AB 3: 45 white, 2 red colonies </ul>
+<ul>kan BA 1: 25 white colonies </ul>
+<ul>kan BA 2: 17 white, 1 red colonies </ul>
+<ul>kan BA 3: 25 white, 1 red colonies </ul>
+<p>Set up overnights with 5 ul kan and 2.5 ul amp per 5ml LB</p>
+</div>
+<div align="left" id='div22' class="hideme">
+<p>July 22, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing a new plasmid with Bfu AB or BA Kan in pSB1A3</span></p>
+<p>Miniprepped overnight tubes (growth in all).</p>
+<p>Digest of pSB1A3 with Bfu kan AB/BA inserts with XbaI and PstI enzymes protocol:</p>
+<ul>plasmid DNA ul </ul>
+<ul>10X NEBuffer 3 2ul </ul>
+<ul>MilliQ H20 12ul </ul>
+<ul>XbaI 1ul </ul>
+<ul>Pst I 1ul </ul>
+<ul> 10X BSA 2ul </ul>
+<ul> Total 20ul </ul>
+<p>Incubated for 1 hour at 37C. </p>
+Insert gel here
+</div>
+<div align="left" id='div23' class="hideme">
+<p>July 23, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing Bfu AB/BA chlor, amp and tet parts</span></p>
+<p>Digest of Bfu Kan AB/BA in pSB1A3 and pSB1C3 with BfuAI protocol:</p>
+<ul>psb1A3 with Bfu Kan AB/BA insert OR psb1C3 with Bfu Kan AB/BA insert 5ul </ul>
+<ul>BfuAI 1ul </ul>
+<ul> NEBuffer 3 10ul </ul>
+<ul> MilliQ H20 84ul </ul>
+<ul> Total 100ul </ul>
+<ul> Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.</ul>
+<ul> Insert gel here </ul>
+<p>Digest of Bsa AB chlor, tet and amp in PSB1C3 with BsaI protocol:</p>
+<ul >psb1C3 with Bsa AB chlor, tet and amp 5ul </ul>
+<ul> BsaI 1ul </ul>
+<ul> NEBuffer 4 10ul </ul>
+<ul> MilliQ H20 84ul </ul>
+<ul> Total 100ul </ul>
+<ul> Incubated at 50C for 1 hour. Heat inactivated at 65C for 20 minutes.</ul>
+<ul> Insert gel here </ul>
+<p>Ligations of Bfu amp AB/BA into pSB1C3 and Bfu chlor and tet AB/BA into pSB1A3 protocol:</p>
+<ul> pSB1A3 or pSB1C3 10ul </ul>
+<ul> antibiotic insert 10ul </ul>
+<ul> 5x ligase buffer 8ul </ul>
+<ul> T4 DNA ligase 1ul </ul>
+<ul> Total 40ul </ul>
+<ul> Incubate at room temperature for one hour. </ul>
+</div>
+<div align="left" id='div26' class="hideme">
+<p>July 26, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing Bfu AB/BA chlor, amp and tet parts</span></p>
+<p>Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 or pSB1C3 ligated with AB and BA antibiotics. Plated on various antibiotic plates.
+</div>
+<div align="left" id='div28' class="hideme">
+<p>July 28, 2010</p><br>
+<span class="h2">Building Parts</span>
+<p><span class="h1">Constructing Bfu AB/BA chlor, amp and tet parts</span></p>
+<p>Overnights Results</p>
+<p>Growth: Bfu AB amp1,2,3; Bfu AB chlor1,2,3; Bfu BA chlor1,2</p>
+<p>No growth: Bfu AB chlor2, Bfu BA chlor3</p>
+<p>Tubes with growth were miniprepped.</p>
 </div>
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