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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>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:</br> | <p>Recipe:</br> | ||
| - | 1μL p1003 (approx. 1ng) | + | <ul>1μL p1003 (approx. 1ng)</ul> |
| - | 2.5μL prA_p1003+ | + | <ul>2.5μL prA_p1003+</ul> |
| - | 2.5μL prB'_p1003- | + | <ul>2.5μL prB'_p1003-</ul> |
| - | 5μL 10X PCR buffer | + | <ul>5μL 10X PCR buffer</ul> |
| - | 1μL 10uM dNTPs | + | <ul>1μL 10uM dNTPs</ul> |
| - | 2μL 50uM MgCl<sub>2</sub> | + | <ul>2μL 50uM MgCl<sub>2</sub></ul> |
| - | 0.5μL Taq polymerase | + | <ul>0.5μL Taq polymerase</ul> |
| - | 35.5μL MilliQ H<sub>2</sub>O</p> | + | <ul>35.5μL MilliQ H<sub>2</sub>O</ul></p> |
<p>Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-. </p> | <p>Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-. </p> | ||
<p>Program: | <p>Program: | ||
| - | 5 min-94<sup>o</sup>C | + | <ul>5 min-94<sup>o</sup>C</ul> |
| - | 45 sec-94<sup>o</sup>C | + | <ul>45 sec-94<sup>o</sup>C</ul> |
| - | 1 min-60<sup>o</sup>C | + | <ul>1 min-60<sup>o</sup>C</ul> |
| - | 1 min-72<sup>o</sup>C | + | <ul>1 min-72<sup>o</sup>C</ul> |
| - | Repeat 2 through 4 35 times | + | <ul>Repeat 2 through 4 35 times</ul> |
| - | 5 min-72<sup>o</sup>C</p> | + | <ul>5 min-72<sup>o</sup>C</ul></p> |
<!--Image of gel performed that day. 5μL of each PCR reaction, 1μL of 10X loading dye and 4μL MilliQ water in a 1% agarose gel --> | <!--Image of gel performed that day. 5μL of each PCR reaction, 1μL of 10X loading dye and 4μL MilliQ water in a 1% agarose gel --> | ||
</div> | </div> | ||
Revision as of 19:54, 8 July 2010
May 2010
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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.
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.
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.
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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. (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
Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-.
Program:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 5 min-72oC
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
