Team:OU-Norman OK/Project/Notebook
From 2013.igem.org
Justin2013 (Talk | contribs) |
Justin2013 (Talk | contribs) |
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<p class = "date">07/2/13</p> | <p class = "date">07/2/13</p> | ||
<div align="center" style="border:1px solid white"><b>PCR of mlsR and Redo of C. sacc and C. beij PCR</b></div></br></br> | <div align="center" style="border:1px solid white"><b>PCR of mlsR and Redo of C. sacc and C. beij PCR</b></div></br></br> | ||
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<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<li>Contents of each tube re suspended in 500μL of PCR dH<sub>2</sub>O</li> | <li>Contents of each tube re suspended in 500μL of PCR dH<sub>2</sub>O</li> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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</table> | </table> | ||
</br> | </br> | ||
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<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
</br> | </br> | ||
- | + | ||
</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
Line 6,285: | Line 6,282: | ||
<p>Procedure performed as outline on pg. 42</p> | <p>Procedure performed as outline on pg. 42</p> | ||
</br> | </br> | ||
- | + | ||
</br> | </br> | ||
<p>16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think</p> | <p>16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think</p> | ||
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</table> | </table> | ||
</br> | </br> | ||
- | + | ||
<p>We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles</p> | <p>We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles</p> | ||
</br> | </br> | ||
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<div align="center" style="border:1px solid white"><b>Gel of puc19 with KIVD Digest (EcoR1 and Pst1)</b></div></br></br> | <div align="center" style="border:1px solid white"><b>Gel of puc19 with KIVD Digest (EcoR1 and Pst1)</b></div></br></br> | ||
</br> | </br> | ||
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<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br> | <div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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</ol> | </ol> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<p class = "date">05/31/13</p> | <p class = "date">05/31/13</p> | ||
<div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin</b></div></br></br> | <div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin</b></div></br></br> | ||
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</br> | </br> | ||
<p>Digestion as preformed on pg. 47</p> | <p>Digestion as preformed on pg. 47</p> | ||
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<div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br> | <div align="center" style="border:1px solid white"><b>Plasmid Isolation of pUC19 from BBa_k273006</b></div></br></br> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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</ol> | </ol> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<p class = "date">05/31/13</p> | <p class = "date">05/31/13</p> | ||
<div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin</b></div></br></br> | <div align="center" style="border:1px solid white"><b>Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin</b></div></br></br> | ||
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</br> | </br> | ||
<p>Digestion as preformed on pg. 47</p> | <p>Digestion as preformed on pg. 47</p> | ||
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<p>Quantification with Nanodrop</p> | <p>Quantification with Nanodrop</p> | ||
</br> | </br> | ||
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</br> | </br> | ||
<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | ||
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</table> | </table> | ||
</br> | </br> | ||
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</br> | </br> | ||
<table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | <table border="1" align="center" style="margin: 1px auto;" style="list-style-type: none; display: inline-block;"> | ||
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</table> | </table> | ||
</br> | </br> | ||
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</br> | </br> | ||
<p>Used wrong kit (Spin mini prep). Suppose to use PCR purification kit. </p>\ | <p>Used wrong kit (Spin mini prep). Suppose to use PCR purification kit. </p>\ | ||
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</ul> | </ul> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<p>Nanodrop Quantification</p> | <p>Nanodrop Quantification</p> | ||
</br> | </br> | ||
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<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<p>4μL PCR dH<sub>2</sub>O</p> | <p>4μL PCR dH<sub>2</sub>O</p> | ||
</br> | </br> | ||
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</br> | </br> | ||
<p>We didn’t get what we were trying to amplify</p> | <p>We didn’t get what we were trying to amplify</p> | ||
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<p>Repeat 30x</p> | <p>Repeat 30x</p> | ||
</br> | </br> | ||
- | + | ||
</br> | </br> | ||
<p>32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin</p> | <p>32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin</p> | ||
Line 8,104: | Line 8,088: | ||
<li>well 4: negative control</li> | <li>well 4: negative control</li> | ||
</ul> | </ul> | ||
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</br> | </br> | ||
<p>The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had</p> | <p>The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had</p> | ||
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<p>2) Nanodrop quantification of plasmid DNA </p> | <p>2) Nanodrop quantification of plasmid DNA </p> | ||
</br> | </br> | ||
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</br> | </br> | ||
<p>3) Digest of pIKM1 and pAN1 with RSA1 | <p>3) Digest of pIKM1 and pAN1 with RSA1 | ||
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</div> | </div> | ||
</br> | </br> | ||
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</br> | </br> | ||
<a href = "#top">Back to top</a> | <a href = "#top">Back to top</a> | ||
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<li>Run gel for 45 minutes-1.5 hours at 85 volts | <li>Run gel for 45 minutes-1.5 hours at 85 volts | ||
</ol> | </ol> | ||
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<p>PCR Reaction Protocol</p> | <p>PCR Reaction Protocol</p> | ||
<ol> | <ol> |
Latest revision as of 04:17, 28 September 2013
09/13/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | Pptb- #9A |
3 | Pptb- #9 B |
4 | Empty |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
09/13/13
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
Pptb- #9A | 2μL | 2μL | 2μL | 2.81μL | 11.19μL | 20μL |
Pptb- #9B | 2μL | 2μL | 2μL | 4.54μL | 9.46μL | 20μL |
09/11/13
QIAprep Spin Miniprep Kit (250)
09/10/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Pptb- #1 | |
3 | pptb- #2 | |
4 | Pptb- #3 | |
5 | Pptb- #4 | |
6 | Pptb- #6 | |
7 | Empty | |
8 | Empty | |
Expected insert size of 125bp
No valid inserts found
Back to top09/11/13
Pptb- #1
Pptb- #2
Pptb- #3
Pptb- #4
Pptb- #6
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
Pptb- #1 | 2μL | 2μL | 2μL | 6.17μL | 9.83μL | 20μL |
Pptb- #2 | 2μL | 2μL | 2μL | 5.79μL | 8.21μL | 20μL |
Pptb- #3 | 2μL | 2μL | 2μL | 7.05μL | 6.95μL | 20μL |
Pptb- #4 | 2μL | 2μL | 2μL | 3.39μL | 10.61μL | 20μL |
Pptb- #5 | 2μL | 2μL | 2μL | 2.95μL | 11.05μL | 20μL |
09/11/13
QIAprep Spin Miniprep Kit (250) Back to top
09/10/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Pptb- #1 | |
3 | pptb- #2 | |
4 | Pptb- #3 | |
5 | Pptb- #4 | |
6 | Pptb- #5 | |
7 | Pptb- #7 | |
8 | Pptb- #14 | |
9 | Pptb- #15 | |
10 | Empty | |
11 | Empty | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #2 | ||
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Pptb- #16 | |
3 | Pptb- #17 | |
4 | Pptb- #18 | |
5 | Pptb- #21 | |
6 | Pptb- #22 | |
7 | Pptb- #23 | |
8 | Pptb- #24 | |
9 | Pptb- #25 | |
10 | Empty | |
11 | Empty | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
09/6/13
3 #17
Pptb- #1
Pptb- #2
Pptb- #3
Pptb- #4
Pptb- #5
Pptb- #7
Pptb- #14
Pptb- #15
Pptb- #16
Pptb- #17
Pptb- #18
Pptb- #21
Pptb- #22
Pptb- #23
Pptb- #24
Pptb- #25
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
Pptb- #1 | 2μL | 2μL | 2μL | 4.78μL | 9.22μL | 20μL |
Pptb- #2 | 2μL | 2μL | 2μL | 4.06μL | 9.94μL | 20μL |
Pptb- #3 | 2μL | 2μL | 2μL | 5.39μL | 8.61μL | 20μL |
Pptb- #4 | 2μL | 2μL | 2μL | 6.71μL | 7.29μL | 20μL |
Pptb- #5 | 2μL | 2μL | 2μL | 4.36μL | 9.64μL | 20μL |
Pptb- #7 | 2μL | 2μL | 2μL | 3.23μL | 10.77μL | 20μL |
Pptb- #14 | 2μL | 2μL | 2μL | 3.91μL | 10.09μL | 20μL |
Pptb- #815 | 2μL | 2μL | 2μL | 4.53μL | 9.47μL | 20μL |
Pptb- #16 | 2μL | 2μL | 2μL | 4.45μL | 9.55μL | 20μL |
Pptb- #17 | 2μL | 2μL | 2μL | 4.02μL | 9.98μL | 20μL |
Pptb- #18 | 2μL | 2μL | 2μL | 2.27μL | 11.73μL | 20μL |
Pptb- #21 | 2μL | 2μL | 2μL | 3.17μL | 10.83μL | 20μL |
Pptb- #22 | 2μL | 2μL | 2μL | 3.43μL | 10.57μL | 20μL |
Pptb- #23 | 2μL | 2μL | 2μL | 3.73μL | 10.27μL | 20μL |
Pptb- #24 | 2μL | 2μL | 2μL | 4.36μL | 9.64μL | 20μL |
Pptb- #25 | 2μL | 2μL | 2μL | 2.63μL | 11.37μL | 20μL |
Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1
Back to top09/6/13
QIAprep Spin Miniprep Kit (250)
09/5/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | Plep #1 |
3 | Empty |
4 | Empty |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
09/5/13
Amount | Component |
---|---|
6.67μL | Plep |
2μL | 10x Buffer |
2μL | EcoR1 |
2μL | Pst1 |
7.33μL | PCR Water |
09/5/13
QIAprep Spin Miniprep Kit (250)
09/4/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | T2 #1 | |
3 | T2 #2 | |
4 | T2 #3 | |
5 | T2 #4 | |
6 | Ter #5 | |
7 | T2 #16 | |
8 | T2 #17 | |
9 | T2 #18 | |
10 | T2 #19 | |
11 | T2 #20 | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #2 | ||
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Pveg2 #1 | |
3 | Pveg2 #2 | |
4 | Pveg2 #3 | |
5 | Pveg2 #4 | |
6 | Pveg2 #5 | |
7 | Pveg2 #16 | |
8 | Pveg2 #17 | |
9 | Pveg2 #18 | |
10 | Pveg2 #19 | |
11 | Pveg2 #20 | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Pptb- #1 | |
3 | Pptb- #2 | |
4 | Pptb- #3 | |
5 | Pptb- #4 | |
6 | Pptb- #5 | |
7 | Pptb- #16 | |
8 | Pptb- #17 | |
9 | Pptb- #18 | |
10 | Pptb- #19 | |
11 | Pptb- #20 | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #2 | ||
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | Plep #1 | |
3 | Plep #2 | |
4 | Plep #3 | |
5 | Plep #4 | |
6 | Plep #5 | |
7 | Plep #16 | |
8 | Plep #17 | |
9 | Plep #18 | |
10 | Plep #19 | |
11 | Plep #20 | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
09/1/13
1) Clos Ori +Ptb- + pSB1A3
1) Clos Ori +Pveg1 + pSB1A3
1) Clos Ori +Pveg2 + pSB1A3
1) Clos Ori +Pliag + pSB1A3
1) mlSR + Plep + pSB1A3
1) mlSR +Terminator2 (4P) + pSB1A3
08/30/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | 2 #8 | |
2 | 2 #10 | |
3 | 2 #12 | |
4 | 3 #15 | |
5 | 3 #17 | |
6 | 4 #1 | |
7 | 4 #3 | |
8 | 5 #1 | |
9 | 6 #1 | |
10 | 6 #3 | |
11 | Ladder (1kb plus) | |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
08/30/13
3 #17
6 #1
6 #3
5 #1
2 #12
3 #15
4 #1
2 #8
2 #10
4 #3
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
3 #17 | 2μL | 2μL | 2μL | 4.06μL | 9.94μL | 20μL |
6 #1 | 2μL | 2μL | 2μL | 5.07μL | 8.93μL | 20μL |
6 #3 | 2μL | 2μL | 2μL | 4.54μL | 9.46μL | 20μL |
5 #1 | 2μL | 2μL | 2μL | 3.08μL | 10.92μL | 20μL |
2 #12 | 2μL | 2μL | 2μL | 6.35μL | 7.65μL | 20μL |
3 #15 | 2μL | 2μL | 2μL | 3.79μL | 10.21μL | 20μL |
4 #1 | 2μL | 2μL | 2μL | 3.41μL | 10.59μL | 20μL |
2 #8 | 2μL | 2μL | 2μL | 4.25μL | 9.75μL | 20μL |
2 #10 | 2μL | 2μL | 2μL | 5.73μL | 8.27μL | 20μL |
4 #3 | 2μL | 2μL | 2μL | 6.03μL | 7.97μL | 20μL |
Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1
Back to top08/30/13
QIAprep Spin Miniprep Kit (250)
08/27/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder (1kb plus) | |
2 | 1 #9 | |
3 | 2 #22 | |
4 | 2 #24 | |
5 | 2 #26 | |
6 | 3 #3 | |
7 | 4 #2 | |
8 | 5 #3 | |
9 | 5 #11 | |
10 | 6 #2 | |
11 | 6 #19 | |
12 | 6 #24 | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
08/26/13
1 #9
ter #18
2 #22
2 #24
2 #26
3 #3
4 #2
5 #3
5 #11
6 #2
6 #19
6 #24
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
1 #9 | 2μL | 2μL | 2μL | 4.21μL | 9.79μL | 20μL |
2 #22 | 2μL | 2μL | 2μL | 6.57μL | 7.43μL | 20μL |
2 #24 | 2μL | 2μL | 2μL | 5.19μL | 8.81μL | 20μL |
2 #26 | 2μL | 2μL | 2μL | 5.03μL | 8.97μL | 20μL |
3 #3 | 2μL | 2μL | 2μL | 4.95μL | 9.05μL | 20μL |
4 #2 | 2μL | 2μL | 2μL | 4.81μL | 9.19μL | 20μL |
5 #3 | 2μL | 2μL | 2μL | 5.91μL | 8.09μL | 20μL |
5 #11 | 2μL | 2μL | 2μL | 4.01μL | 9.99μL | 20μL |
6 #2 | 2μL | 2μL | 2μL | 6.35μL | 7.65μL | 20μL |
6 #19 | 2μL | 2μL | 2μL | 3.54μL | 10.46μL | 20μL |
6 #24 | 2μL | 2μL | 2μL | 4.99μL | 9.01μL | 20μL |
08/27/13
QUIAPREP/Spin Miniprep Kit < (250)
08/26/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | ter #6 |
3 | ter #18 |
4 | ter #24 |
5 | Adh6 #8 |
6 | Adh6 #14 |
7 | Adh6 #15 |
8 | Empty |
08/26/13
ter #6
ter #18
ter #24
Adh6 #8
Adh6 #14
Adh6 #15
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
ter #6 | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
ter #18 | 2μL | 2μL | 2μL | 4.63μL | 9.37μL | 20μL |
ter #24 | 2μL | 2μL | 2μL | 1.59μL | 12.41μL | 20μL |
Adh6 #8 | 2μL | 2μL | 2μL | 1.67μL | 12.33μL | 20μL |
Adh6 #14 | 2μL | 2μL | 2μL | 5.24μL | 11.76μL | 20μL |
Adh6 #15 | 2μL | 2μL | 2μL | 1.52μL | 12.48μL | 20μL |
08/26/13
QIAprep Spin Miniprep Kit (250)
08/22/13
- Transform 4μL of ligation product into 100μL aliquots of competent E. coli cells
- Incubate on ice for 30 minutes
- Heat shock in water bath for 60 seconds
- Incubate on ice for 5 minutes
- Add 600μL of psi broth
- Incubate at 37°C for 2 hours at 200 rpm
- Make necessary dilutions and plate out
08/22/13
1) Clos Ori +Pveg2 (20G) + pSB1A3
1) Clos Ori +Plep (20E) + pSB1A3
1) Clos Ori +Pveg1 (23B) + pSB1A3
1) Clos Ori +Pliag (20A) + pSB1A3
1) mlSR +Terminator1 (6D) + pSB1A3
1) mlSR +Terminator2 (4P) + pSB1A3
Amount | Sample |
---|---|
2μL | Upstream Part |
2μL | Downstream Part |
2μL | Destination Backbone |
2μL | 10x T4 DNA Ligase Buffer |
0.5μL | T4 DNA Ligase |
10μL | PCR Water |
Incubate at room temperature for 10 minutes, then at 80°C for 20 minutes to inactivate EcoR1
Back to top08/15/13
Clos Ori
mlSR
Pliag
Pveg1
Plep
Pveg2
Terminator
Terminator
pSB1A3
Sample | EcoR1 | Pst1 | Xba1 | Spe1 | 100x Bovine Serum Albumin | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|---|---|---|
Clos Ori | 1μL | 1μL | - | - | 1μL | 5μL | 5.7μL | 36.3μL | 50μL |
mlSR | 1μL | 1μL | - | - | 1μL | 5μL | 9.08μL | 32.92μL | 50μL |
Pliag | - | - | 1μL | 1μL | 1μL | 5μL | 5.7μL | 39.99μL | 50μL |
Pveg1 | - | - | 1μL | 1μL | 1μL | 5μL | 2.78μL | 39.22μL | 50μL |
Pveg2 | - | - | 1μL | 1μL | 1μL | 5μL | 1.67μL | 40.33μL | 50μL |
Plep | - | - | 1μL | 1μL | 1μL | 5μL | 3.08μL | 38.92μL | 50μL |
Terminator 6D | - | - | 1μL | 1μL | 1μL | 5μL | 1.15μL | 40.85μL | 50μL |
Temrinator 4P | - | - | 1μL | 1μL | 1μL | 5μL | 0.95μL | 41.05μL | 50μL |
pSB1A3 | 1μL | 1μL | - | - | 1μL | 5μL | 16μL | 26μL | 50μL |
08/21/13
Previous attempt produced a lot of extraneous artifacts.
Primer concentration was too high and so was the template concentrations
Used Grad program previously used to run PCR machine
50°C to 60°V used 4 and 0
Back to top08/21/13
The concentrations of pSB1A3 is low. Therefore, the SPRI based size selection was conducted on it once more. The gel results are displayed below.
08/21/13
- Thaw competent Top10 cell of E. coli from -80°C (100μL aliquots)
- Add 20μL of ligation product to Top 10 E. coli
- Incubate on ice for 30 minutes
- Heat shock in water bath for exactly 60 seconds
- Incubate on ice for 5 minutes
- Add 600μL of psi broth
- Incubate at 37°C for 2 hours at 200 rpm
- Make dilutions using 90μL of psi broth: 1:10, 1:100, 1:1000
- Plate out 25μL undiluted and diluted solutions onto a petri dish
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | pSB1A3 |
3 | Empty |
4 | Empty |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/21/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/21/13
Use the instructions labeled “left side size slection.” This kit will hopefully remove the primer dimer from our product. Remove the primer dimer from the backbone PCR clean-up.
Back to top08/21/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder (1kb plus) |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/21/13
Sample G in the primer matrix will be used as a template for this particular PCR
Label | Amount | Component |
---|---|---|
PCR # 1 | 3μL | SBprep-3P-1 |
1μL | SBprep-2Ea | |
2.0μL | pSB1A3 | |
94μL | PCR Supermix High Fidelity | |
PCR #3 | 3μL | SBprep-3P-1 |
1μL | SBprep-2Ea | |
2.0μL | pSB1C3 | |
PCR #2 | 3μL | SBprep-3P-1 |
1μL | SBprep-2Ea | |
2.0μL | pSB1K3 | |
94μL | PCR Supermix High Fidelity |
08/20/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | E |
3 | G |
4 | Empty |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/15/13
Use the two concentrations of E and G on 20-August
08/20/13
After discussing the results with Dr. Wawrik, it has been decided to cut the number of cycles down to thirty, and to increase the template used by two.
The best concentrations of primers to be used are indicated by the results of the primer matrix on letters E and G. F appeared to have been a probable pipetting error.
List of reactants and amount for primer matrix PCR optimization
Label | Amount | Component |
---|---|---|
E | 0.5μL | SBprep-3P-1 (1:2 dilutions) use 1μL |
1.5μL | SBprep-2Ea | |
1.0μL | Template | |
46.5μL | PCR Supermix High Fidelity | |
G | 1.5μL | SBprep-3P-1 |
0.5μL | SBprep-2Ea (1:2 dilutions) use 1μL | |
1.0μL | Template | |
46.5μL | PCR Supermix High Fidelity |
08/19/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | Adh6 #19 |
3 | Adh6 #3 |
4 | Ter #5 |
5 | Ter #23 |
6 | Ter #26 |
7 | Ter #9 |
8 | Empty |
08/14/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/14/13
Although the concentrations are good, the purity is still in question. It is an improvement on the PCR from 13-August
One possibility is that the original stocks used as templates in the PCR may not be pure enough. Therefore, a gel will be run on these original stocks
Back to top08/14/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/14/13
QIAquick PCR purification kit (250) is used before viewing the post clean-up product on a gel
Back to top08/14/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | 50°C |
3 | |
4 | |
5 | 60°C |
6 | Empty |
7 | Empty |
8 | Empty |
What happened here? What is in the lanes above the comb?
Many have some product and we will use the forward and reverse primers to get the whole gene, if present
08/14/13
Row 1 | |
---|---|
Lane Number (1kb plus) | Sample |
1 | Ladder |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/14/13
The following stock are to be used for backbone amplification in the PCR procedure illustrated on 14-August
08/14/13
Fragment 1: 340bp – 125.6ng/μL
Fragment 1: 660bp – 66.3ng/μL
Fragment 1: 300bp – 231.7ng/μL
Use 1μL of fragment 3 = 231.7ng/μL
Fragment 2:
Fragment 3:
Added to a 1.5mL epi tube and diluted to 200μL
Back to top08/14/13
PCR #1 | |
---|---|
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1A3 (1ng/μL) |
PCR #2 | |
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1C3 (1ng/μL) |
PCR #3 | |
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1K3 (1ng/μL) |
08/14/13
Diluting concentrations of pSB1A3, pSB1C3, pSB1K3 to 1ng/μL
Quantification of current backbones to confirm concentrations before conducting dilution
pSB1A3
pSB1C3
pSB1K3
Back to top08/13/13
The concentrations are good, however the purity of the product is not. Actions taken to remedy this problem are to run another PCR using 1ng/μL of template and running the third step of the PCR cycle at 68°C for 5 minutes.
08/13/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | pSB1A3 |
3 | pSB1C3 |
4 | pSB1K3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/13/13
The three backbones were cleaned up using QIAquick PCR Purification Kit (250) so that the next gel will much more clearly display the backbone.
Back to top08/13/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | pSB1K3 |
3 | pSB1C3 |
4 | pSB1A3 |
5 | Empty |
6 | Empty |
7 | Empty |
8 | Empty |
08/13/13
QUIAPREP/Spin Miniprep Kit < (250)
Cells were centrifuged from an inoculated broth
None of the concentrations obtained are high enough to work with. Must go about finding a more efficient plasmid extraction for these samples.
Back to top08/13/13
PCR #1 | |
---|---|
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1C3 (43ng/μL) |
PCR #2 | |
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1C3 (74.8ng/μL) |
PCR #3 | |
Amount (μL) | Component |
100 | PCR Supermix High Fidelity |
0.7 | SB-prep-3P-1 |
0.7 | SB-prep-2Ea |
0.6 | pSB1C3 (55.1ng/μL) |
08/14/13
All Fragment 1’s, lanes 8 and 9 of Fragment 2, and all Fragment
08/13/13
08/9/13
Row #1 | ||
---|---|---|
Lane Number | Sample | Temperature (°C) |
1 | Ladder | |
2 | Fragment 1 | 50 |
3 | Fragment 1 | |
4 | Fragment 1 | |
5 | Fragment 1 | 60 |
6 | Fragment 2 | 50 |
7 | Fragment 2 | |
8 | Fragment 2 | |
9 | Fragment 2 | 60 |
10 | Fragment 3 | 50 |
11 | Fragment 3 | |
12 | Fragment 3 | |
13 | Fragment 3 | 60 |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Based on 2% agarose gel, 115V for about 30 minutes
Going to PCR the whole gene to see if I can reduce non-specific products in fragment #2
08/8/13
Preformed gradient PCR for all 3 BktB fragments
Undiluted template was used
08/7/13
- Thaw competent 100μL aliquot of E. coli cells from -80°C freezer
- Add 20μL of ligation poduct to Top10 E. coli
- Incubate on ice for 30 minutes
- Heat shock in water bath for 60 seconds
- Incubate on ice for 5 minutes
- Add 600μL of psi broth
- Incubate at 37°C for 2 hours at 200 rpm
- Make a dilution series of 1:10, 1:100, 1:1000, and 1:10000
08/7/13
Into 1.5mL microcentrifuge tubes
Amount (μL) | Component |
---|---|
20 | Heat killed digest of pSB1C3 |
20 | Heat killed digest of Adh6 |
1.0 | T4 Ligase |
5.0 | 10X T4 Ligase Buffer |
4.0 | Nuclease Free PCR Water |
50 | Total |
Incubate on bench for 45 minutes
Back to top08/9/13
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
ter #23 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL |
ter #7 | 1μL | 1μL | 1μL | 1.5μL | 15.5μL | 20μL |
ter #26 | 1μL | 1μL | 1μL | 2μL | 15μL | 20μL |
ter #5 | 1μL | 1μL | 1μL | 2μL | 15μL | 20μL |
pSB1C3 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL |
08/9/13
08/7/13
Adh1 #5
Adh1 #19
Adh1 #24
Adh1 #4
Adh1 #13
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
Adh1 #5 | 2μL | 2μL | 2μL | 1.5μL | 12.5μL | 20μL |
Adh1 #19 | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL |
Adh1 #24 | 2μL | 2μL | 2μL | 2μL | 12μL | 20μL |
Adh1 #4 | 2μL | 2μL | 2μL | 2μL | 12μL | 20μL |
Adh1 #13 | 2μL | 2μL | 2μL | 1μL | 13μL | 20μL |
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
Adh6 #5 | 1μL | 1μL | 1μL | 5.0μL | 12.0μL | 20μL |
Adh6 #19 | 1μL | 1μL | 1μL | 2.0μL | 15.0μL | 20μL |
Adh6 #3 | 1μL | 1μL | 1μL | 3.0μL | 14.0μL | 20μL |
Adh6 #15 | 1μL | 1μL | 1μL | 3.0μL | 14.0μL | 20μL |
pSB1C3 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL |
08/7/13
Using 2% agarose in the gel
Gel 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | #16-6D |
3 | #16-23B |
4 | #9-4P |
5 | #9-6D |
6 | #3-23B |
7 | #14-20E |
8 | Empty |
Gel 2 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | #15-20A |
3 | #7-20G |
4 | #7-4D |
5 | #14-20A |
6 | #10-4P |
7 | #11-20E |
8 | Empty |
Samples display a possible insert, and will be sent off for sequencing
3-23B, 16-23B, 14-20E, 15-20A, 16-6D, 14-20A, and 10-4P
Back to top08/7/13
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
#16-6D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#16-23B | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#9-4P | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#9-6D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#3-23B | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#14-20E | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#15-20A | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#7-20G | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#7-4D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#14-20A | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#10-4P | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
#11-20E | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL |
08/7/13
QIAPrep Spin Miniprep Kit (250)
The gene’s origin is unknown, and will be found upon sequencing
07/18/13
07/17/13
07/11/13
07/25/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder | |
2 | Aadc- #6 | |
3 | Aadc- #23 | |
4 | Aadc+ #20 | |
5 | thl- #4 | |
6 | Aadc+ #15 | |
7 | Aadc+ #2 | |
8 | thl+ #17 | |
9 | thl+ #3 | |
10 | thl+ #10 | |
11 | thl- #14 | |
12 | thl- #21 | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #2 | ||
Lane Number | Sample | |
1 | Ladder | |
2 | Empty | |
3 | Empty | |
4 | Empty | |
5 | Empty | |
6 | Empty | |
7 | Ladder | |
8 | Ptb- #5 | |
9 | Ptb+ #9 | |
10 | Ptb+ #1 | |
11 | Ptb+ #26 | |
12 | Ptb- #18 | |
13 | Ptb- #8 | |
14 | Empty | |
15 | Empty | |
16 | Empty | |
08/02/13
QIAPrep Spin Miniprep Kit (250)
08/02/13
#2 Aadc+ 500ng (1μL⁄85.9ng) = 6.0μL
#15 Aadc+ 500ng (1μL⁄117.5ng) = 4.5μL
#20 Aadc+ 500ng (1μL⁄92.1ng) = 5.5μL
#23 Aadc- 500ng (1μL⁄184.8ng) = 3.0μL
#6 Aadc- 500ng (1μL⁄218.1ng) = 2.5μL
#17 thl+ 500ng (1μL⁄100.0ng) = 5.0μL
#3 thl+ 500ng (1μL⁄84.4ng) = 6.0μL
#10 thl+ 500ng (1μL⁄90.4ng) = 5.5μL
#14 thl- 500ng (1μL⁄85.7ng) = 6.0μL
#21 thl- 500ng (1μL⁄89.1ng) = 5.5μL
#4 thl- 500ng (1μL⁄131.8ng) = 4.0μL
#9 Ptb+ 500ng (1μL⁄81.4ng) = 6.0μL
#1 Ptb+ 500ng (1μL⁄124.2ng) = 4.0μL
#26 Ptb+ 500ng (1μL⁄206.7ng) = 2.5μL
#18 Ptb- 500ng (1μL⁄103.7ng) = 5.0μL
#8 Ptb- 500ng (1μL⁄150.1ng) = 3.5μL
#5 Ptb- 500ng (1μL⁄216.0ng) = 2.5μL
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
#2 Aadc+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL |
#15 Aadc+ | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL |
#20 Aadc+ | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL |
#23 Aadc- | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL |
#6 Aadc- | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
#17 thl+ | 2μL | 2μL | 2μL | 5.0μL | 9.0μL | 20μL |
#3 thl+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL |
#10 thl+ | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL |
#14 thl- | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL |
#21 thl- | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL |
#4 thl- | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
#9 Ptb+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL |
#1 Ptb+ | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
#26 Ptb+ | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
#18 Ptb- | 2μL | 2μL | 2μL | 5.0μL | 9.0μL | 20μL |
#8 Ptb- | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL |
#5 Ptb- | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
08/02/13
QIAPrep Miniprep Kit (250)
08/01/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | 3:23B |
3 | 10:4P |
4 | 12:20G |
5 | 9:6D |
6 | 16:6D |
7 | 8:20G |
8 | 11:20E |
9 | 14:20E |
10 | 15:20A |
11 | 14:20A |
12 | 9:4P |
13 | 16:23B |
14 | 9:20G |
15 | 7:20G |
08/1/13
#16-6D 500ng (1μL⁄108.6ng) = 4.5μL
#14-20E 500ng (1μL⁄235.2ng) = 2.0μL
#12-20G 500ng (1μL⁄135.1ng) = 3.5μL
#8-20G 500ng (1μL⁄192.3ng) = 2.5μL
#14-20A 500ng (1μL⁄156.5ng) = 3.0μL
#9-6D 500ng (1μL⁄`99.7ng) = 2.5μL
#10-4P 500ng (1μL⁄131.8ng) = 4.0μL
#7-20G 500ng (1μL⁄232.8ng) = 2.0μL
#3-23B 500ng (1μL⁄90.0ng) = 5.5μL
#16-23B 500ng (1μL⁄123.5ng) = 4.0μL
#9-4P 500ng (1μL⁄145.2ng) = 3.5μL
#9-20G 500ng (1μL⁄106.8ng) = 4.5μL
#15-20A 500ng (1μL⁄150.1ng) = 3.5μL
#11-20E 500ng (1μL⁄126.2ng) = 4.0μL
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
#16-6D | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL |
#14-20E | 2μL | 2μL | 2μL | 2.0μL | 12.0μL | 20μL |
#12-20G | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL |
#8-20G | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
#14-20A | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL |
#9-6D | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
#10-4P | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
#7-20G | 2μL | 2μL | 2μL | 2.0μL | 12.0μL | 20μL |
#3-23B | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL |
#16-23B | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
#9-4P | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL |
#9-20G | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL |
#15-20A | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL |
#11-20E | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
08/1/13
QIAPrep Spin Miniprep Kit (250)
08/01/13
To make 40mL of ampicillin 1x antibiotic
- 2.0g ampicillin 1000x stock from 4°C refrigerator
- Pour the amipicillin ino a 50mL falcon
- Fill flacon wih dH2O to 40mL mark
- Use 10mL syringe with puradisc 25mm filter to dispense 10mL of ampicillin 1x into four 10mL falcons (leaves alternative stocks if one is contaminated)
- (Agar: 10g)
- Bacto Tryptone: 16g
- Yeast Extract: 10g
- Sodium Chloride: 5g
- Distilled Water: 1000mL
- R. eutropha = 27 July_ 16.9
- R. eutropha = 24 July_ 25
- R. eutropha = 24 July_ 38.9
- Power Soil Extraction = 25 July_ 28.3
- Phenol Chloroform Extraction = 25 July_ 637
- Mix: 98μL
- Template: 2μL
- Total: 100μL
- 2x Master Mix: 250μL
- Forward Primer: 3μL
- Reverse Primer: 3μL
- PCR dH2O: 244μL
- Total: 500μL
- Forward + SDM1 Reverse
- 340 base pairs
- SDM1 Forward + SDM2 Revers
- 660 base pairs
- SDM2 Forward + Reverse
- 300 base pairs
- Re suspend cells in 500μL 1x STE buffer
- Vortex until mixture is homogenous
- Add 50μL of 10% Sodium-dodecyl sulfate (SDS)
- IF cells insufficiently lysed, 2 minutes in bead beater
- Add 500μL of Chloroform:Phenol pH 8
- Extract bottom layer (2 phases present)
- Minimize time the chloroform is out of fridge
- Vortex
- Centrifuge for 30 seconds
- 14000 rpm
- Transfer top phase to 1.5mL microcentrifuge tube
- Don't take up white precipitant from interphase of solution
- Add 500μL of isoamyl acetate (CIAA 24:1) to top phase
- Vortex
- Centrifuge for 30 seconds
- 14000 rpm
- Transfer top phase to 1.5mL microcentrifuge tube
- Don't take up white precipitant from interphase of solution
- REPEAT STEPS 9-12
- Note: Keep track of amount finally transfered in step 12
- Add (2/10) volume (amount of top phase) of 3M Sodium Acetate
- Add (7/10) volume (step 14 amount) of Isopropanol
- Note: Mix solutions with pipette
- Centrifuge for 25 minutes
- 14000 rpm
- Dispose of liquid portion
- Don't disturb invisible pellet
- Dry pellet on heat block at 60°C for 10 minutes
- Leave caps open for evaporation of isopropanol
- Re suspend pellet in 50μL of distilled water
- Backbone: 2μL
- EcoR1: 1μL
- Pst1: 1μL
- 10x Buffer: 2μL
- Water: 14μL
- Total: 20μL
- gBloeu Resuspension: 10μL
- EcoR1: 1μL
- Pst1: 1μL
- 10x Buffer: 2μL
- Water: 6μL
- Total: 20μL
- pSB1C3: 2μL
- Promoter: 13μL
- Buffer: 2μL
- Ligase: 1μL
- Water: 2μL
- Total: 20μL
- BBa_K148012 Pveg
- BBa_K823000 P?
- BBa_K823002 P?
- BBa_K823003 Pveg
- BBa_K090504 ?
- BBa_B1006
- 39 base pairs
- 2013 p3w6D in C3
- BBa_B1002
- 34 base pairs
- 2013 p5w4D in Ak3
- For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
- 1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
- Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
- Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
- Vortex
- 50μL of 10% SDS added to each tube
- 500μL of TE saturated phenol pH8 added to each tube
- Vortex well and centrifuge for 30 seconds
- Aqueous recovered
- Phenol extraction
- Steps 7-9 repeated
- 500μL chloroform added to each tube and vortexed
- Aqueous discarded
- Chloroform extraction repeated
- 400μL split into each of four tubes
- 100μL of 3M NaOH added to each tube
- 350μL isopropanol added to each tube
- Centrifuge at 14000 rpm for 25 minutes
- Liquid discarded
- Tubes places on 80°C heating block for 15 minutes
- Contents of each tube re suspended in 500μL of PCR dH2O Back to top
- Undiluted
- 1:10
- 1:100
- 100mL in bottles
- 20mL in vials
- Clean glass vial with acetone
- In aerobic Chamber:
- Chemwipes
- Sterile Needles
- Bring into chamber
- Heavy duty aluminum foil to work over
- Glass cutter
- File
- Cycle chamber airlock; twice
- Clean gloves and chamber with 1N HCl (x2)
- ???????????????????
- Use sterile syringe to add 0.5mL media to inner vial
- Mix with syringe
- Add 0.5 of inoculums to each of the two media vials
- Transfer an inoculum from these vials to a second vial to get one heavy and one light inoculum
- Spun at 12000 g for 20 minutes
- Discarded supernatant and re suspended in 15mL of TFBl
- Incubated on ice for 60 minutes
- Centrifuged at 12000 g for 20 minutes
- Re suspend cells in 500μL 1x STE buffer
- Vortex
- Add 1/10 volume of 10% SDS
- 2 minutes in bead beater
- Add 500μL TE saturated phenol pH 8
- Be sure to go down far enough in bottle to reach phenol
- Put directly back in fridge when done
- Vortex
- Centrifuge for 30 seconds
- Recover aqueous phase
- Don’t recover white material at interphase
- Throw away
- Add 500μL Chloroform: Isoamyl acetate
- Vortex
- Centrifuge for 30 seconds
- Recover and throw away aqueous phase
- Repeat Steps 8-12
- Add 2/10 volumes 3M sodium acetate
- Add 7/10 volumes isopropanol
- Mix well
- Centrifuge for 25 minutes
- Remove liquid and don’t disturb invisible pellet
- Dry pellet at 60°C in heat block for 10 minutes
- Leave caps off to allow isopropanol to evaporate
- Re suspend pellet in 50μL of dH2
- Re suspend cells in 500μL 1x STE buffer
- Vortex
- Add 1/10 volume of 10% SDS
- 2 minutes in bead beater
- Add 500μL TE saturated phenol pH 8
- Be sure to go down far enough in bottle to reach phenol
- Put directly back in fridge when done
- Vortex
- Centrifuge for 30 seconds
- Recover aqueous phase
- Don’t recover white material at interphase
- Throw away
- Add 500μL Chloroform: Isoamyl acetate
- Vortex
- Centrifuge for 30 seconds
- Recover and throw away aqueous phase
- Repeat Steps 8-12
- Add 2/10 volumes 3M sodium acetate
- Add 7/10 volumes isopropanol
- Mix well
- Centrifuge for 25 minutes
- Remove liquid and don’t disturb invisible pellet
- Dry pellet at 60°C in heat block for 10 minutes
- Leave caps off to allow isopropanol to evaporate
- Re suspend pellet in 50μL of dH2
- Insert, Vector: 21μL
- Ligase Buffer: 5μL
- Ligase: 3μ:L
- 5μL 10x Buffer
- 2μL ligase (quick)
- ?μL vector
- ?μL insert
- Up to 50μL dH2
- Turn on heating block
- Add equal amounts of phenol and whatever volume DNA is in (20μL)
- Phenol : Pipette below top surface
- Vortex
- Centrifuge for 30 seconds
- Two layers appear
- Protein is inactive and at interphase
- Pipette out top layer of both and combine aqueous
- Need to get rid of phenol because it absorbs same wavelength as does water and interferes with enzyme activity
- Add 50μL of CIAA 24:1
- Removes Phenol
- Vortex
- Centrifuge for 30 seconds
- Remove top layer (aqueous)
- Leave a little behind so not to contaminate aqueous phase with bottom layer
- Add 50μL of CIAA 24:1
- Vortex
- Centrifuge for 30 seconds
- Remove top layer (aqueous)
- Leave a little behind so not to contaminate aqueous phase with bottom layer
- Add 1/10 volume sodium acetate 3M pH 5.2
- Add 7/10 volume (total) of isopropanol
- Vortex
- Centrifuge for 20 minutes at 14,000 rpm
- Dispose of liquid, leaving pellet inside tube
- Add 50μL of 10% ethanol to get rid of isopropanol
- Centrifuge for 30 seconds
- Dispose of liquid
- Use heat block to dry
- 60°C for 15 minutes
- Leave tube open
- Add 10μL of PCR water to re suspend pellet
- Use 2μL to quantify via nandrop
- Add 1μL Ligase (quick)
- Add 1μL 10x Buffer = 10μL reaction
- Let reaction sit at room temperature for 2 hours
- 95°C for 2 minutes
- 95°C for 30 seconds
- 55°C for 30 seconds
- 68°C for 3 minutes
- 69°C for 10 minutes
- 10μL of each NYP
- 60μL of dH2O
- Dilutions
- 1:10
- 1:100
- 1:1000
- 95°C for 45 seconds
- 52°C for 45 seconds
- 72°C for 2 minutes
- 500mL dH2
- 15g TSB mix
- 1g glucose
- Adjust pH to 7.3
- Place sponge stopper in place
- Open silver valve and black valve
- Set degassing station to 20 psi
- Switch to nitrogen and run for 30 seconds to flush oxygen out of head space
- Place largest needle into media and turn on spin located on stir-plate, then set time for 45 minutes
- Disinfect top of bottle with alchol wipe
- Add sterile wipe filter with new needle
- Need steady stream of bubbles
- Add 0.1mL Resasrin (will be purple until autoclaved)
- Post autoclaved
- Pink color signifies media has been exposed to oxygen
- No color in media signifies media is anaerobic
- Oxygen scavenger
- If black precipitant forms after autoclave and before transfer into media bottles, then too much oxygen present.
- Use acid washed vials
- Place stoppers in ice and water to shrink pores
- Flush bottles and vials with nitrogen
- Place 35mL in each using automated pipette
- Angle stopper with needle (gas still within)
- Leave for 10 seconds
- Bring needle out of bottle and push stopper in
- Crimp top
- Add 15mL to vials
- Flush head space
- Put regular needle in and flushing needle in
- Flush for 3 minutes
- Pull both needles out at same times so you don’t put pressure on tubes
- Switch degassing station to vacuum
- Alternate between vacuum and pressure 10 times
- Let it settle back to position before going back to pressure
- Make sure all needles are in black stopper
- Tubes now have 20 psi and poke with needle to vent
- Turn off tank
- Autoclave immediately
- 95°C for 45 seconds
- 52°C for 45 seconds
- 72°C for 2 minutes
- well 1: 1:10 dilution of template
- well 2: 1:100 dilution
- well 3: 1:1000 dilution
- well 4: negative control
- 95°C for 2 minutes
- 95°C for 30 seconds
- 55°C for 30 seconds
- 68°C for 3 minutes
- 68°C for 10 minutes
- pSB1C3
- RSA1 and Xba1
- 2μL of Invitrogen buffer
- pSB1C3
- RSA1 and Xba1
- 2μL of Fermentas buffer
- pSB1C3
- RSA1 and Xba1
- 1μL of Fermentas buffer and 1μL of Invitrogen buffer
- 5μL Plasmid
- 2μL RSA1
- 2μL 10x Buffer
- 11μL PCR Water
- 5μL Plasmid
- 2μL RE1(XbaI)
- 2μL RE2(Hind 3 or RSA1
- 2μL 10x Buffer
- 9μL PCR Water
- 0.8g of agarose
- 40mL of TAE 1x
- Only a 1:100 dilution was used since our DNA template had a concentration of 164.6 ng/μL, which is a good value to use for genomic DNA.
- A temperature gradient for the annealing step was setup where different columns in thermocycler are a different temperature during the annealing process.
- Highlight stage of interest (in this case; annealing)
- Selection "options"
- Select "Show Gradient"
- well 1:Ladder
- well 2:10 Dilution
- well 3:100 Dilution
- well 4:1000 Dilution
- well 5:10000 Dilution
- well 6:Control
- Click icon with ND-1000 on computer
- Click "Nucleic Acids"
- Wipe off pedestal with chemwipe
- Load 3°L DI water to initialize and click blank
- Click "Measure" to verify flat line
- Load 3°L of sample
- Click "Measure"
- Click "Print" screen after sample ID has been typed in
- In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
- Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
- Run flask bottom under water to cool agarose
- Pour into gel rig with comb inserted
- Let gel cool until opaque
- Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
- Remove comb slowly
- Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
- Load samples into wells after 1 kb DNA ladder is loaded into far left lane
- well 1:Ladder
- well 2:pSB1C3 EcoR1
- well 3:pSB1C3 EcoR1 Pst1
- well 4:pSB1C3 Pst1
- well 5:pSB1A3 EcoR1
- well 6:pSB1A3 EcoR1 Pst1
- well 7:pSB1A3 Pst1
- well 8:pSB1C3 UNDIGESTED
- Run gel for 45 minutes-1.5 hours at 85 volts
- Run gel for 45 minutes-1.5 hours at 85 volts
PCR Reaction Protocol
- Combine the following
- 150μL 2x master mix (polymerase,buffer)
- 1μL forward primer
- 1μL reverse primer
- 148μL DI water (PCR water, UV prior to use)
- Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
- In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
- in fifth tube, only put in step 1 solution as a negative control
Regular PCR Cycle
- 95°C for 45 seconds
- 55°C for 1 minute
- 75°C for 1.5 minutes
- These three steps are cycled 35 times
03/12/13
Preformed the following restriction digest of pSB1A3 and pSB1C3.
500ng DNA in 20μL
pSB1A3 [DNA] = 135.8ng/μL
pSB1C3 [DNA] = 74.7ng/μL
500ng x μL/135.8μg = 3.68μL
500ng x μL/74.7μg = 6.69μL
Sample EcoRI PstI 10X Buffer DNA PCR Water TOTAL pSB1A3 2μL 0μL 2μL 4μL 12μL 20μL pSB1A3 2μL 2μL 2μL 4μL 10μL 20μL pSB1C3 2μL 0μL 2μL 7μL 9μL 20μL pSB1C3 2μL 2μL 2μL 7μL 7μL 20μL 03/8/13
Nanodrop DNA QuanitificationProtocol
- Open Nanodrop 7000 V.6.0
- Select Nucleic Acid
- Blank via 3μL of PCR water
- Verify 0ng/&#;L DNA in PCR water
- Load 3μL of sample
- Record DNA concentration in ng/μL
- Print Screen
pSB1K3
p34KM
pIKM1
pSB1A3
pSB1C3
Back to top
02/13/13
Colonies were counted
1:1000 dilution of AmpicillinR pSB1A3 wasn't properly plated
Dilution Plasmid Colony Count Cells/μg DNA 1:1000 pSB1C3 58 8.24e6 1:1000 pSB1K3 79 1.12e7 1:1000 p34KM 157 2.22e7 1:100 pSB1A3 1521 2.16e7 Note: 1:100 estimates by counting colonies in 1/3 of plate and multiplying by 3
Back to top02/12/13
Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight
Back to top02/13/13
pSB1C3
Dilution 1:1000
58 Colonies
Back to top02/11/13
Transforming Competent Cells
We transformed TOP10 chemically competent cells using plasmids.
Resistance Plasmid ID Original Concentration (ng/μL) Kanamycin pSB1K3 62 Kanamycin p34KM 40 Chloramphenicol pSB1C3 43 Ampicillin pSB1A3 75 Protocol
- TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
- 100ng of plasmid were added to cells
- Cells were placed on ice for 20 minutes
- Cells were transformed to a 42°C waterbath for 60 seconds
- After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
- Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
- Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
- 50mL of each dilution was plated on an LB + Antibiotic plate
- Plates were incubated at 37°C overnight
Plasmids were diluted to 20μL of 10μg/μL
p34KM
pSB1C3
pSB1A3
pSB1K3
Back to topBuffers for Preparing Competent E. coli
02/4/13
TFB1: pH: 5.8/ Sterile Filter
Chemicals Concentration (mM) RbCl 100 MnCl2 50 Potassium Acetate 30 CaCl2 10 Glycerol 15% by Weight TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter
Chemicals Concentrations (mM) MOPS 10 RbCl 10 CaCl2 75 Glycerol 15% by Weight TFB1 Chemicals Mass (g) RbCl 3.02965 MnCl2 1.56996 Potassium Acetate 0.74392 CaCl2 0.27806 Glycerol 37.5463 TFB2 Chemicals Mass (g) MOPS 0.53398 RbCl 0.30225 CaCl2 2.88320 Glycerol 37.5457 Making Antibiotic Stocks
02/1/13
Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.
Make antibiotic plates with the following specs.
Antibiotic Concentration (µg/mL) Color Code Ampicillin 100 Orange Chloramphenicol 35 Green Kanamycin 50 Red Tetracycline 15 Yellow Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.
Stocks of antibiotics are made at the following concentrations
Ampicillin 100 mg/mL Chloramphenicol 35 mg/mL Kanamycin 30 mg/mL Tetracycline 15 mg/mL 50mL of stock were prepared and split into several 15mL tubes
Stocks should be stored in the refrigerator at 4°C
Back to top01/25/13
Restreaked TOP10 cells on LB plates for isolation of single colonies.
Back to top01/23/13
Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator
Back to top01/18/13
Poured LB agar plates
Back to top01/16/13
Making Agar Plates
LB agar is used to grow our stocks of Escherichia coli
Recipe: Per 1000 mL- 10g Bacto Tryptone
- 5g Yeast Extract
- 10g Sodium Chloride (NaCl)
- 15g Agarose
Mix Components in 1L of dH2O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.
Back to top - Combine the following
Ampicillin 1000x = 50ng/mL
50ng/mL x 40mL = 2.0g Ampicillin 1000x
Back to top07/31/13
Used 2XYT liquid medium instead of LB to increase carbon amount in order to enhance growth of cultures.
-3mL of 2XYT with Chloramphenicol antibiotic
-Pellet of culture
Incubated at 37°C with shaking at 220rpm
Back to top07/31/13
Fill 1L jar with 500mL of dH2O. Pour components and magnetic stirring rod into jar. Fill jar with another 500mL of dH2O until the shoulder of the jar is reached.
Autoclave for 20 minutes at slow exhaust (Setting #2)
Back to top07/31/13
Run a gel with 3μL of dye and 5μL of DNA extract for each of our R. eutropha extracts.
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder | |
2 | Phenol Chloroform (27 July) | |
3 | R. eutropha 1 (24 July) | |
4 | R. eutropha 2 (24 July) | |
5 | Power Soil (25 July) | |
6 | Phenol Chloroform (25 July) | |
7 | Empty | |
8 | Empty | |
NOTE: Lane six shows chewed up DNA by nucleases
Disposed all samples except for Power Soil because it has the highest efficacy
07/30/13
Plasmid purification of top 10 cells transformed with ligation of pSB1C3 and Ca promoters (Ptb-RBS, Ptb+RBS, thl+RBS, thl-RBS, Aadc+RBS, AaDc-RBS)
07/30/13
Used QIAPrep Spin Miniprep Kit (250)
07/27/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | #14-20A |
3 | #17-20G |
4 | #14-20E |
5 | #11-20E |
07/27/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | #10-4p |
3 | #14-20E |
4 | #14-20A |
5 | #7-20G |
6 | #11-20E |
7 | #16-20E |
8 | #9-4P |
9 | #15-20A |
07/11/13
500ng in 20μL with 2μL for each digest
#11-20E 500ng (1μL⁄58.1ng) = 8.5μL
#9-4P 500ng (1μL⁄40.2ng) = 12.5μL
#14-20A 500ng (1μL⁄43.5ng) = 12.5μL
#7-20G 500ng (1μL⁄59.4ng) = 12.5μL
#14-20E 500ng (1μL⁄37.5ng) = 12.5μL
#10-4P 500ng (1μL⁄43.6ng) = 12.5μL
#15-20A 500ng (1μL⁄51.6ng) = 12.5μL
#16-23B 500ng (1μL⁄52.5ng) = 12.5μL
Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
#11-20E | 2μL | 2μL | 2μL | 8.5μL | 5.5μL | 20μL |
#9-4P | 2μL | 2μL | 2μL | 12.5μL | 1.5μL | 20μL |
#14-20A | 2μL | 2μL | 2μL | 11.5μL | 2.5μL | 20μL |
#7-20G | 2μL | 2μL | 2μL | 8.5μL | 5.5μL | 20μL |
#14-20E | 2μL | 2μL | 2μL | 13.5μL | 0.5μL | 20μL |
#10-4P | 2μL | 2μL | 2μL | 11.5μL | 2.5μL | 20μL |
#15-20A | 2μL | 2μL | 2μL | 9.5μL | 4.5μL | 20μL |
#16-23B | 2μL | 2μL | 2μL | 9.5μL | 4.5μL | 20μL |
07/26/13
07/26/13
Row #1 | ||
---|---|---|
Lane Number | Sample | |
1 | Ladder | |
2 | R. eutropha extract (27 July) | |
3 | R. eutropha 1 (24 July) | |
4 | R. eutropha 2 (24 July) | |
5 | Power Soil (25 July) | |
6 | Phenol Chloroform (25 July) | |
7 | Empty | |
8 | Empty | |
07/26/13
Repeated gel with larger loading of DNA. Removing only loaded 5μL of dye pls products. Wanted to see if bands appear brighter.
Row #1 | ||
---|---|---|
Lane Number | Sample | Temperature (°c) |
1 | Kb Plus Ladder | |
2 | Piece 1 | 50 |
3 | Piece 1 | 52.7 |
4 | Piece 1 | 54.5 |
5 | Piece 1 | 56.4 |
6 | Piece 1 | 60 |
7 | Piece 2 | 50 |
8 | Piece 2 | 52.7 |
9 | Piece 2 | 54.5 |
10 | Piece 2 | 56.4 |
11 | Piece 2 | 60 |
12 | Ladder | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #1 | ||
---|---|---|
Lane Number | Sample | Temperature (°c) |
1 | Kb Plus Ladder | |
2 | Piece 3 | 50 |
3 | Piece 3 | 52.7 |
4 | Piece 3 | 54.5 |
5 | Piece 3 | 56.4 |
6 | Piece 3 | 60 |
7 | Whole gene | 50 |
8 | Whole gene | 52.7 |
9 | Whole gene | 54.5 |
10 | Whole gene | 56.4 |
11 | Whole gene | 60 |
12 | Ladder | |
13 | Empty | |
14 | Empty | |
15 | Empty | |
16 | Empty | |
07/11/13
Wanted to identify which extracts may have DNA present
Previous gradient seems to have produced a PCR product for 3 piece. Used temperature 55°C, since this is the predicted optimal temperature for the SDM2 forward and the BktB_Rev primers
Templates used:
07/25/13
Row #1 | ||
---|---|---|
Lane Number | Sample | Temperature (°c) |
1 | Kb Plus Ladder | |
2 | Forward + SDM1 Reverse | 50 |
3 | Forward + SDM1 Reverse | 52.7 |
4 | Forward + SDM1 Reverse | 54.5 |
5 | Forward + SDM1 Reverse | 56.4 |
6 | Forward + SDM1 Reverse | 60 |
7 | SDM1 Forward + SDM2 Reverse | 50 |
8 | SDM1 Forward + SDM2 Forward | 52.7 |
9 | SDM1 Forward + SDM2 Reverse | 54.5 |
10 | SDM1 Forward + SDM2 Reverse | 56.4 |
11 | SDM2 Forward + Reverse | 60 |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | EMpty | |
16 | Empty | |
Row #2 | ||
Lane Number | Sample | Temperature (°C) |
1 | Ladder | |
2 | SDM2 Forward + Reverse | 50 |
3 | SDM2 Forward + Reverse | 52.7 |
4 | SDM2 Forward + Reverse | 54.5 |
5 | SDM2 Forward + Reverse | 56.4 |
6 | SDM2 Forward + Reverse | 60 |
7 | Forward + Reverse | 50 |
8 | Forward + Forward | 52.7 |
9 | Forward + Reverse | 54.5 |
10 | Forward + Reverse | 56.4 |
11 | Forward + Reverse | 60 |
12 | Empty | |
13 | Empty | |
14 | Empty | |
15 | Empty | |
16 | Empty | |
07/25/13
If DNA template is the problem with the PCR, then one of the following procedures may correct it.
1) Phenol Chloroform extraction using the protocol from July 24, 2013 with the following modification: 2μL of lysozyme added in the 3rd step with the 10% SDS, to encourage membrane disolution
2)Using the MoBio Power Soil DNA extraction kit and included protocol.
07/25/13
Most plates had growth. Plates are going to be incubated at 37°c for another night. Tomorrow a library will be made.
Back to top07/26/13
Colonies were larger. Transformant libraries were made of Ca promoters in addition to ter, Adh6, and C. cell Adh. Plates were parafilmed and placed in 37°C incubator to prevent drying out over the weekend. Monday, plates will be streaked, Tuesday, plasmids will be extracted, and Wednesday, plasmids will be sent for sequencing.
Back to top07/25/13
Repeated PCR of BktB using primers for sie-directed mutagenesis
Also included one PCR series using the Forward and Revers primers for the whole gene
Used only undiluted templates
Temperature gradient was selected to identify correct temperature for each pair of primers (assuming template is present)
Used wells:
1: T = 50°:c
4: T = 52.7°:c
6: T = 54.5°:c
8: T = 56.4°:c
12: T = 60°:c
07/25/13
Row #1 | ||
---|---|---|
Lane Number | Sample | Dilution |
1 | Empty | |
2 | Kb plus ladder | |
3 | Forward + SDM1 Reverse | 1:10 |
4 | Forward + SDM1 Reverse | 1:100 |
5 | Forward + SDM1 Reverse | 1:1000 |
6 | Forward + SDM1 Reverse | 1:10000 |
7 | SDM1 Forward + SDM2 Reverse | 1:10 |
8 | SDM1 Forward + SDM2 Forward | 1:100 |
9 | SDM1 Forward + SDM2 Reverse | 1:1000 |
10 | SDM1 Forward + SDM2 Reverse | 1:10000 |
11 | SDM2 Forward + Reverse | 1:10 |
12 | SDM2 Forward + Reverse | 1:100 |
13 | SDM2 Forward + Reverse | 1:1000 |
14 | SDM@ Forward + Reverse | 1:10000 |
15 | Kb plus Ladder | |
16 | Empty | |
No PCR products.
Will run a temperature gradient for each sample to see if temperature is affecting enzyme activity
Will preform Power Soil DNA Extraction and bead beaten with phenol: chloroform extraction and isopropanol precipitation to see if template is the problem.
Back to top07/24/13
Into each tube:
Dilutions used: 1:10, 1:100, 1:1000, 1:10000
Mix:
PCR Primers Used:
07/24/13
07/24/13
Preformed as outlined on February 11, 2013
-Last time we used 2μL for transformation this time since our ligations will contain a maximum of 100ng of insert (recommended amount for transformation of top 10 cells), we are going to use the entire ligation volume (20μL)
Back to top07/23/13
Component | Amount |
---|---|
Beef Extract | 3g |
Peptone | 5g |
Agar | 15g |
Distilled Water | 1000mL |
Pour components and one magnetic mixer into empty jar. Fill jar with distilled water to 1000mL mark.
Autoclave at setting number "2" for 20 minutes at slow exhaust
Back to top07/23/13
Component | Amount |
---|---|
2X Master Mix | 125μL |
Forward Primer | 4μL |
Reverse Primer | 4μL |
PCR Water | 117μL |
TOTAL | 250μL |
1 Reaction 50μL = 49μL Mix + 1μL Template
07/23/13
500ng DNA for each reaction
500ng (1μL⁄39.7ng) = 12.5 μL Cc DNA
500ng (1μL⁄36.0ng) = 14 μL Cs DNA
Component | Cc | Cs | pSB1C3 |
---|---|---|---|
10X Buffer | 2μL | 2μL | 2μL |
EcoR1 | 2μL | 2μL | 2μL |
Pst1 | 2μL | 2μL | 2μL |
DNA | 12.5μL | 14μL | 2.5μL |
PCR Water | 1.5μL | 0μL | 11.5μL |
Total | 20μL | 20μL | 20μL |
500ng (1μL⁄202ng) = 2.5μL pSB1C3 backbone
Incubate at 37°C water bath for 45 minutes
Back to top07/11/13
1. Digest
A. In 1 tube contain
B. In each 6 tubes combine
Incubate in 37°C water bath for 1 hour
Clean with QIAquick PCR Purification Kit
Nano drop
![HTML tutorial](https://static.igem.org/mediawiki/2013/9/91/107.png)
Aadc quantification appears to be good with a reading of 1.9ng/μL, but is the lowest limit of detection for the nano drop 1000, so we can't be sure
Ligation
Since the vector is 31.7 ng/μL and the protocol calls for 50ng of DNA and the concentration of insert might be 1.5 to 2.0ng/μL due to the need of 20ng.
Incubate at room temperature overnight
Back to top07/23/13
Row #1 | |||
---|---|---|---|
Lane Number | Sample | Dilution | Temperature(μ) |
1 | Ladder | ||
2 | Ca AdhE | 1:100 | 53 |
3 | Ca AdhE | 1:100 | 50 |
4 | Ca AdhE | 1:100 | 56 |
5 | Ca AdhE | 1:100 | 60 |
6 | Ca AdhE | 1:10 | 50 |
7 | Ca AdhE | 1:10 | 53 |
8 | Ca AdhE | 1:10 | 56 |
9 | Ca AdhE | 1:10 | 60 |
10 | Ca AdhE | 1:1000 | 50 |
11 | Ca AdhE | 1:1000 | 53 |
12 | Ca AdhE | 1:1000 | 56 |
13 | Ca AdhE | 1:1000 | 60 |
14 | Empty | ||
15 | Empty | ||
16 | Empty | ||
Row #2 | |||
1 | Ladder | ||
2 | Ca AdhE1 | 1:100 | 50 |
3 | Ca AdhE1 | 1:10 | 50 |
4 | Ca AdhE1 | 1:100 | 53 |
5 | Ca AdhE1 | 1:100 | 50 |
6 | Ca AdhE1 | 1:100 | 56 |
7 | Ca AdhE1 | 1:10 | 53 |
8 | Ca AdhE1 | 1:10 | 56 |
9 | Ca AdhE1 | 1:10 | 60 |
10 | Ca AdhE1 | 1:1000 | 50 |
11 | Ca AdhE1 | 1:1000 | 53 |
12 | Ca AdhE1 | 1:1000 | 56 |
13 | Ca AdhE1 | 1:1000 | 60 |
14 | Empty | ||
15 | Empty | ||
16 | Empty | ||
07/22/13
Sample | Dilution | 50° | 53° | 56° | 60° |
---|---|---|---|---|---|
Empty | |||||
Ca AdhE | 1:10 | ✓ | ✓ | ✓ | ✓ |
Ca AdhE | 1:100 | ✓ | ✓ | ✓ | ✓ |
Ca AdhE | 1:1000 | ✓ | ✓ | ✓ | ✓ |
Empty | |||||
Ca AdhE1 | 1:10 | ✓ | ✓ | ✓ | ✓ |
Ca AdhE1 | 1:100 | ✓ | ✓ | ✓ | ✓ |
Ca AdhE1 | 1:1000 | ✓ | ✓ | ✓ | ✓ |
![HTML tutorial](https://static.igem.org/mediawiki/2013/0/01/105.png)
07/22/13
Used Qia Quick Prep Plasmid Extraction Kit
AdhE and AdhE1 are supposed to be located on pSOL plasmid using pSOL as template (1:10, 1:100, 1:1000)
07/22/13
Used Qia Quick Prep Plasmid Extraction Kit
07/11/13
Row #1 | |
---|---|
Well # | Sample |
1 | Ladder |
2 | mlsR 5 |
3 | mlsR 9 |
4 | mlsR 10 |
5 | mlsR 12 |
6 | mlsR 22 |
7 | mlsR 24 |
07/22/13
EcoR1 and Pst1 Dual Digest of mlsR colonies
5, 9, 10, 12, 22, 24
500ng (1μL⁄112.2ng) = 4.45 μL → 4.5 μL
500ng (1μL⁄124.5ng) = 4.02 μL → 4.0 μL
500ng (1μL⁄117.7ng) = 4.25 μL → 4.5 μL
500ng (1μL⁄162.0ng) = 3.09 μL → 3.0 μL
500ng (1μL⁄188.7ng) = 2.65 μL → 2.5 μL
500ng (1μL⁄206.4ng) = 2.42 μL → 2.5 μL
Colony Number | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total |
---|---|---|---|---|---|---|
5 | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL |
9 | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL |
10 | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL |
12 | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL |
22 | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
24 | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |
Incubated at 37°L for 45 minutes
Transformed to 80°L heat block for 1 hour
Back to top07/22/13
Transforming the following parts from the iGEM distribution for use in shuttle vector
BBa_B1006 from 2013 Plate 3, Well 6D (in C3)
BBa_Bl003 from 2013 Plate 3, Well 4P (in C3)
Punctured wells with pipette tip
Re suspended DNA in 10μL of PCR water
1μL of DNA added to 50μL of competent cells
100μL of cells Used 2μL of DNA
Incubated on ice for 30 minutes
Heat shocked cells for 1 minutes
Incubated on ice for 5 minutes
Add 600μL of Psi broth
Incubated at 37°C with shaking for 2 hours
Also Xformed
Plated 90μL of undiluted 1:10, 1:100, 1:1000 of BBa_B1006 and BBa_B1003
Back to top07/22/13
07/22/13
mlsR library colonies 5, 9, 10, 12, 22, 24
Extracted using Qiagen plasmid prep kit
07/19/13
Preformed PCR to amplify Adh out of C. cellulolyticum
07/19/13
Choose lower temperature (50°C) for annealing.
Still have primer dimer, or primers may be sitting down at another place on the genomic DNA
Back to top07/19/13
No colonies have grown on the plates incubated with transformed Top 10 cells with pSB1C3 and various promoters plated on July 16 2013. We did a nanodrop quantification of the ligationproducts.
While the 260/280 ratios seem high, Dr. wawrick says that they aren’t troubling. However, the amount of DNA quantified is unrealistic. Since, we only started with 100ng of insert and 200ng of vector. We ran a gel to see if what we quantified is DNA and not RNA.
Well 1-Ladder
Well 2-Aadc – RBS
Well 3- thl + RBS
From this gel, it doesn’t look like we actually have any DNA (although there may be some really faint bands near the bottom). This means that the ligation didn’t actually occur (or we would have seen a band at 2000bp + 125-75bp). However, if the ligation didn’t work we would still expect to see 2 bands. One is the size of the vector and the other is the size of the insert. Perhaps, something went wrong in the phenol/chloroform extraction step.
Back to top07/19/13
Transferred six colonies from library to individual plates. Numbers 5, 9, 10, 12, 22, and 24 from library.
PCR Purification of July 18, 2013 Cs
Back to top07/18/13
Row 1 | |||
---|---|---|---|
Lane Number | Sample | Dilution | Temperature (°C) |
1 | Ladder | ||
2 | Cs | Undiluted | 55 |
3 | Cs | Undiluted | 62 |
4 | Cs | Undiluted | 65 |
5 | Cs | 1:10 | 55 |
6 | Cs | 1:10 | 62 |
7 | Cs | 1:10 | 65 |
8-16 | Empty | ||
Row 2 | |||
Lane Number | Sample | Dilution | Temperature (°C) |
1 | Ladder | ||
2 | Cb | Undiluted | 55 |
3 | Cb | Undiluted | 62 |
4 | Cb | Undiluted | 65 |
5 | Cb | 1:10 | 55 |
6 | Cb | 1:10 | 62 |
Cb | Cs | 1:10 | 65 |
8-16 | Empty | ||
07/18/13
Row 1 | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | pSB1C3 + mlsR1 |
3 | pSB1C3 + mlsR7 |
4 | pSB1C3 + mlsR13 |
5 | pSB1C3 + mlsR13 |
6 | pSB1C3 + mlsR21 |
7 | pSB1C3 + mlsR23 |
8 | pSB1C3 + mlsR26 |
9 | puc19 + KIVD |
07/18/13
500ng of plasmid for digest
puc19 + KIVD
pSB1C3 + mlsR21
pSB1C3 + mlsR13
pSB1C3 + mlsR1
pSB1C3 + mlsR7
pSB1C3 + mlsR23
pSB1C3 + mlsR26
Gene | EcoR1 | Pst1 | 10x Buffer | DNA | PCR Water | Total |
---|---|---|---|---|---|---|
KIVD | 2μL | 2μL | 2μL | 1μL | 13μL | |
mlsR21 | 2μL | 2μL | 2μL | 5μL | 9μL | |
mlsR13 | 2μL | 2μL | 2μL | 6μL | 8μL | |
mlsR1 | 2μL | 2μL | 2μL | 5μL | 9μL | |
mlsR7 | 2μL | 2μL | 2μL | 7.5μL | 6.5μL | |
mlsR23 | 2μL | 2μL | 2μL | 6μL | 8μL | |
mlsR26 | 2μL | 2μL | 2μL | 6μL | 8μL | |
07/18/13
07/17/13
07/10/13
The following sequences have been verified:
Clos Ori BBa_J238001
Pthl BBa_J238002
Paadc BBa_J238003
Ptb BBa_J238004
mlsR BBa_J238005
Use Terminators for test testing
Long artificial terminator (T>90%)
Small artificial terminator (T = 85%)
07/10/13
Procedure followed as on pg. 50
Row 1 | ||||
---|---|---|---|---|
Lane | Sample | Dilution | Temperature (°C) | |
1 | Ladder | |||
2 | C. sacc | 1:10 | 50.8 | |
3 | C. sacc | 1:100 | 50.8 | |
4 | C. sacc | 1:1000 | 50.8 | |
5 | mlsR | 1:10 | 50.8 | |
6 | mlsR | 1:100 | 50.8 | |
7 | mlsR | 1:1000 | 50.8 | |
8 | C beij | 1:10 | 50.8 | |
9 | C beij | 1:100 | 50.8 | |
10 | empty | |||
11 | empty | |||
12 | empty | |||
13 | empty | |||
14 | empty | |||
15 | empty | |||
16 | empty | |||
Row 2 | ||||
1 | Ladder | |||
2 | C. sacc | 1:10 | 52.8 | |
3 | C. sacc | 1:100 | 52.8 | |
4 | C. sacc | 1:1000 | 52.8 | |
5 | mlsR | 1:10 | 52.8 | |
6 | mlsR | 1:100 | 52.8 | |
7 | mlsR | 1:1000 | 52.8 | |
8 | C beij | 1:10 | 52.8 | |
9 | C beij | 1:100 | 52.8 | |
10 | empty | |||
11 | empty | |||
12 | empty | |||
13 | empty | |||
14 | empty | |||
15 | empty | |||
16 | empty | |||
Row 3 | ||||
1 | Ladder | |||
2 | C. beij | 1:10 | 54.1 | |
3 | C. beij | 1:100 | 54.1 | |
4 | mlsR | 1:10 | 54.1 | |
5 | mlsR | 1:100 | 54.1 | |
6 | mlsR | 1:1000 | 54.1 | |
7 | C. sacc | 1:10 | 54.1 | |
8 | C. sacc | 1:100 | 54.1 | |
9 | C. sacc | 1:1000 | 54.1 | |
10 | empty | |||
11 | empty | |||
12 | empty | |||
13 | empty | |||
14 | empty | |||
15 | empty | |||
16 | empty | |||
Row 2 | ||||
1 | Ladder | |||
2 | C. beij | 1:10 | 58.8 | |
3 | C. beij | 1:100 | 58.8 | |
4 | mlsR | 1:10 | 58.8 | |
5 | mlsR | 1:100 | 58.8 | |
6 | mlsR | 1:1000 | 58.8 | |
7 | C. sacc | 1:10 | 58.8 | |
8 | C. sacc | 1:100 | 58.8 | |
9 | C. sacc | 1:1000 | 58.8 | |
10 | empty | |||
11 | empty | |||
12 | empty | |||
13 | empty | |||
14 | empty | |||
15 | empty | |||
16 | empty | |||
07/9/13
Procedure followed as on pg. 50
Back to top07/9/13
Column Number | Temperature (°C) |
---|---|
3 | 50 |
5 | 52.8 |
6 | 54.1 |
10 | 58.8 |
Lane Number | Sample |
---|---|
1 | C sacc 1:10 |
2 | C sacc 1:100 |
3 | C sacc 1:1000 |
4 | mlsR 1:10 |
5 | mlsR 1:100 |
6 | mlsR 1:1000 |
7 | C beij 1:10 |
8 | C beij 1:100 |
Vf Preparation
Component | Amount |
---|---|
2x Mastermix | 125μL |
Forward | 2μL |
Reverse | 2μL |
PCR dH2O | 121μL |
Total | 250μL |
07/3/13
Row 1 | ||
---|---|---|
Lane | Sample | |
1 | Ladder | |
2 | C. sacc 1:10 | |
3 | C. sacc 1:100 | |
4 | C. sacc 1:1000 | |
5 | C. sacc 1:10000 | |
6 | Negative | |
7 | mlsR 1:10 | |
8 | mlsR 1:100 | |
9 | mlsR 1:1000 | |
10 | mlsR 1:10000 | |
11 | Negative | |
12 | ladder | |
13 | empty | |
14 | empty | |
15 | empty | |
16 | empty | |
Row 2 | ||
1 | Ladder | |
2 | C. sacc 1:10 | |
3 | C. sacc 1:100 | |
4 | C. sacc 1:1000 | |
5 | C. sacc 1:10000 | |
6 | Negative | |
7 | C. beij 1:10 | |
8 | C. beij 1:100 | |
9 | C. beij 1:1000 | |
10 | C. beij 1:10000 | |
11 | empty | |
12 | empty | |
13 | empty | |
14 | empty | |
15 | empty | |
16 | empty | |
07/2/13
07/2/13
Due to previous low extraction efficiency
Attempted to extract higher [DNA]
07/2/13
Lane | Sample | |
---|---|---|
1 | Ladder | |
2 | C. sacc Undiluted | |
3 | C. sacc 1:10 | |
4 | C. sacc 1:100 | |
5 | Negative | |
6 | C. cell undiluted | |
7 | C. cell 1:10 | |
8 | C. cell 1:100 | |
Lane | Sample | |
---|---|---|
1 | Ladder | |
2 | Negative Control | |
3 | 1:10 Diluted C. beij | |
4 | C. beij 1:100 | |
5 | C. beij 1:1000 | |
6 | C. beij 1:10000 | |
7 | Empty | |
8 | Empty | |
07/1/13
16S gel of C. acetobutylicum, R. eutropha, C. sacchaolyticum, and C. beijerinki to confirm wheather or not we have what we think we have
16S Gel | |
---|---|
Lane Number | Sample |
1 | Ladder |
2 | C. beijerinki |
3 | R. eutropha |
4 | C. sacchrolyticum |
C. acetobutylicum | |
06/28/13
1 part loading dye
2 parts 40% glycerol
2 parts PCR DI water
Back to top06/27/13
Procedure performed as outline on pg. 42
16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think
PCR Solution | |
---|---|
Component | Amount (μL) |
2x PCR Mastermix | 350 |
Forward primer (27F) | Reverse primer (1525R |
PCR water | 344 |
Total | 700 |
Place 1μL template into each tube
Negative control won’t have a template
Row Number | Colum Number | Sample |
---|---|---|
3 | 7 | Negative Control |
4 | 4 | C. beijerinki |
4 | 5 | C. beijerinki 1:10 |
4 | 6 | C. beijerinki 1:100 |
4 | 7 | C. sacchrolyticum |
4 | 8 | C. sacchrolyticum 1:10 |
4 | 9 | C. sacchrolyticum 1:100 |
5 | 4 | C. acetobutylicum |
5 | 5 | C. acetobutylicum 1:10 |
5 | 6 | C. acetobutylicum 1:100 |
5 | 7 | R. eutropha |
5 | 8 | R. eutropha 1:10 |
5 | 9 | R. eutropha 1:100 |
Gel of PCR product | ||
---|---|---|
Row Number | Colum Number | Sample |
1 | 1 | Ladder |
1 | 2 | C. beijerinki |
1 | 3 | C. beijerinki 1:10 |
1 | 4 | C. beijerinki 1:100 |
1 | 5 | C. sacchrolyticum |
1 | 6 | C. sacchrolyticum 1:10 |
1 | 7 | C. sacchrolyticum 1:100 |
2 | 1 | Ladder |
2 | 2 | C. acetobutylicum |
2 | 3 | C. acetobutylicum 1:10 |
2 | 4 | C. acetobutylicum 1:100 |
2 | 5 | R. eutropha |
2 | 6 | R. eutropha 1:10 |
2 | 7 | R. eutropha 1:100 |
2 | 8 | Negative Control |
We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles
Back to top06/25/13
C. cellulolyticum may be grown at 37°L
VM medium to reduce precicpitation
AEM: 77, 2727-2733 (2011)
Modified ? (per liter) | |
---|---|
Component | Amount |
KH2PO4 | 1g |
KH2PO4 | 3.4g |
Urea | 2.14g |
MgCl2 - 6H2O | 1g |
CaCl2 - 2H2O | 0.15g|
FeSO2 - 6H2 | 1.25g |
MOPS 3-(N-morpholino)propanesulfuric acid | 10g |
Rezazurin | 2mg |
Vitamin solution | 10mL |
Yeast Extract | 2g |
Oligoelement solution | 1mL |
Cysteine – HCl | 1g |
Cellobiose | 5.1345 |
100x Vitamin Solution | |
---|---|
Component | Amount|
Biotin | 0.08μM |
Pyridoxamine | 0.02μM |
Cyanocobalamine | 0.001μM |
p-aminobenzoic acid | 0.15μM |
Thiamine | 0.9μM |
L-alaning | 0.22μM |
1000x Oligoelement Solution (per liter) | |
---|---|
Component | Amount |
FeSO4 - 7H2O | 5g |
ZnSO4 - 7H2O | 1.44g |
MnSO4 - 7H2O | 1.12g |
CuSO4 - 5H2O | 0.25g |
Na2B4O7 | 0.2g |
(Mo)7(NH4)6O24 - 4H2O | 1g |
NiCl2 | 0.04g |
CoCl2 | 0.02g |
HBO3 | 0.03g |
Na2SeO3 | 0.02g |
10M HCl | 50mL |
VM Medium placement
06/25/13
06/24/13
Media components were added to a 2000mL Erlenmeyer flask
0.1mL of 1g/L Rezarin was added to the medium and was degassed under N2 for 45 minutes
Back to top06/21/13
C. acetobutylicum will grow on defined media:
AEM 50:1238 (1985) or the following
Dr. Tanner’s recipe of C .acetobutylicum (per liter) | |
---|---|
Component | Amount |
Yeast Extract | 1g |
Glucose/Dextrose | 20g |
RST minerals | 10mL |
RST vitamins | 10mL |
RST metals | 5mL |
RST Minerals (per liter) | |
---|---|
Component | Amount |
NaCl | 80g |
NH4Cl | 100g |
KCl | 10g |
KH2PO4 | 10g |
MgSO4 - 7H2O | 20g |
CaCl2 - 2H2O | 4g |
RST Minerals (per liter) | |
---|---|
Component | Amount |
Nitriloacetic Acid | 2g |
MnSO4 - H2O | 1g |
Fe(NH4)2(SO4)2 - 6H2O | 0.8g |
CoCl2 - 6H2O | 0.2g |
2nSO4 - 7H2 | 0.2g |
CuCl2 - 2H2O | 0.02g |
NiCl2 - 6H2O | 0.02g |
Na2MoO4 - 2H2O | 0.02 |
Na2SeO4 | 0.02g |
Na2WO4 | 0.02g |
RST Vitamins | ||
---|---|---|
Components | Amount | Light Sensitivity (Y/N) |
Pyridoxine HCl | 10mg | Y |
Thiamine HCl | 5mg | Y |
Riboflavin | 5mg | Y |
Calcium Pantothenate | 5mg | Y |
Thioctic Acid | 5mg | N |
p-Aminobenzoic Acid | 5mg | Y |
Nicotinic Acid | 5mg | N |
B12 = Cyanocobalamine | 5mg | N |
Biotin | 2mg | N |
Folic Acid | 2mg | Y |
Mercaptopethane Sulfuric Acid | 10mg | N |
06/17/13
Suspended a unit sized inoculums of pAN1 containing DH5 E. coli in LB with chloramphenicol resistance.
Incubated overnight at 37°C and at 220 rpm for 2.5 hours
One culture has an OD600 of 0.38 and another is 0.475
06/14/13
06/14/13
Component | Amount (μL) |
---|---|
EcoR1 | 1 |
Pst1-Hf | 1 |
10x Buffer | 2 |
DNA | 2.5 (500ng) |
PCR H2 | 13.5 |
Total | 20 |
Place in 37°C water bath for 45 minutes
Back to top06/13/13
Row 1 | ||
---|---|---|
Well Number | Sample | Temperature (°C) |
1 | Ladder | |
2 | AdhE1 | 50.8 |
3 | AdhE1 | 52.8 |
4 | AdhE1 | 54.1 |
5 | AdhE1 | 58.8 |
Row 2 | ||
---|---|---|
Well Number | Sample | Temperature (°C) |
1 | Ladder | |
2 | AdhE | 50.8 |
3 | AdhE | 52.8 |
4 | AdhE | 54.1 |
5 | AdhE | 58.8 |
The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).
AdhE = 2589 base pairs
AdhE1 = 2577 base pairs Back to top06/12/13
Temperature gradient and dilution up to 1:10000
Column Number | Temperature (°C) |
---|---|
3 | 50.8 |
5 | 52.8 |
6 | 54.1 |
10 | 58.8 |
06/12/13
06/10/13
Testing our primers using the template prepared on June/4
Forward primer used (AdhE)
Forward primer sequence
Reverse primer used (AdhE)
Reverse primer Sequence
Forward primer used (AdhE1)
Forward primer sequence
Reverse primer used (AdhE1)
Reverse primer sequence
Preparation of solution 1:
Into two separate 1.5mL centrifuge tubes
Component | Amount (μL) |
---|---|
PCR DI Water | 146 |
2x PCR Mastermix | 150 |
Forward primer | 2 |
Reverse primer | 2 |
Total | 300 |
50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)
Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000
1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1
The 5th PCR tube containing solution 1 will serve as negative control
Back to top06/4/13
Place pxy plates in glove box to degrass
Extracted Ca DNA
05/31/13
Digestion as preformed on pg. 47
Gel of Digestion | |
---|---|
Row 1 | |
Well Number | Sample |
1 | Ladder |
2 | 1 |
3 | 3 |
4 | 5 |
5 | 8 |
6 | 11 |
7 | 12 |
Row 2 | |
Well Number | Sample |
1 | Ladder |
2 | 16 |
3 | 18 |
4 | 20 |
5 | 22 |
6 | 24 |
Wells 2 and 3 like like what we need.
Sequencing primers are in the process of being made to comfirm
Row 2 | ||
---|---|---|
Well Number | Sample | Temperature (°C) |
1 | Ladder | |
2 | AdhE | 50.8 |
3 | AdhE | 52.8 |
4 | AdhE | 54.1 |
5 | AdhE | 58.8 |
The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).
AdhE = 2589 base pairs
AdhE1 = 2577 base pairs Back to top06/12/13
Temperature gradient and dilution up to 1:10000
Column Number | Temperature (°C) |
---|---|
3 | 50.8 |
5 | 52.8 |
6 | 54.1 |
10 | 58.8 |
06/12/13
06/10/13
Testing our primers using the template prepared on June/4
Forward primer used (AdhE)
Forward primer sequence
Reverse primer used (AdhE)
Reverse primer Sequence
Forward primer used (AdhE1)
Forward primer sequence
Reverse primer used (AdhE1)
Reverse primer sequence
Preparation of solution 1:
Into two separate 1.5mL centrifuge tubes
Component | Amount (μL) |
---|---|
PCR DI Water | 146 |
2x PCR Mastermix | 150 |
Forward primer | 2 |
Reverse primer | 2 |
Total | 300 |
50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)
Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000
1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1
The 5th PCR tube containing solution 1 will serve as negative control
Back to top06/4/13
Place pxy plates in glove box to degrass
Extracted Ca DNA
05/31/13
Digestion as preformed on pg. 47
Gel of Digestion | |
---|---|
Row 1 | |
Well Number | Sample |
1 | Ladder |
2 | 1 |
3 | 3 |
4 | 5 |
5 | 8 |
6 | 11 |
7 | 12 |
Row 2 | |
Well Number | Sample |
1 | Ladder |
2 | 16 |
3 | 18 |
4 | 20 |
5 | 22 |
6 | 24 |
Wells 2 and 3 like like what we need.
Sequencing primers are in the process of being made to comfirm
Back to top05/30/13
Purified six tubes of DNA (plasmid)
Quantification with Nanodrop
Digestion | |
---|---|
Component | Amount (μL) |
EcoR1 | 2 |
Pst1 | 2 |
10x Buffer | 1 |
5 | |
Incubate in 37°C water bath for 1 hour
Load all 10μL onto gel
Gel of Digestion | |
---|---|
Well Number | Sample |
1 | Ladder |
2 | Screen #3 |
3 | Screen #6 |
4 | Screen #10 |
5 | Screen #19 |
6 | Screen #19 |
7 | Screen #26 |
Note: Screen # corresponds to quantification Id
Since our insert is about 700 base pairs and our vector is about 2000 base pairs. Well 2 looks like what we want. We are going to re streak colonies isolated from plate with screen #3 , make a freezer stock, and take more colonies from screen #10
Freezer stocks of Top 10, pAN1, pIKM1, and p34KM were made by suspending cells in 1mL of 10% glycerol.
Back to top05/29/13
Made 80 stocks of C. acetobutylicum (2mL of culture into 2mL of glycerol)
Streaked three more Top 10 pSB1C3 and Clostridial Origin plates
Back to top05/28/13
Inoculated cultures of C. acetobutylicum and a transfer was made
Six plates (LB + Chloramphenicol) were streaked with transformation of pSB1C3 and Clostridial Origin in Top 10 E. coli cells in preparation for plasmid extraction.
Back to top05/23/13
b>After 824: possible growth Back to top
05/22/13
Procedure as preformed on pg. 38
Gel of pSB1C3 and Insert Digestion | |
---|---|
Well Number | Sample |
1 | Ladder |
2 | pSB1C3 |
3 | Clostridial Origin |
Gel of pSB1C3 and Insert Digestion after Quanitification | |
---|---|
Well Number | Sample |
1 | Ladder |
2 | pSB1C3 |
3 | Clostridial Origin |
Ligation:
Let reaction sit on bench for 90 minutes
OR
For overnight ligation: 14°C water bath
Back to top05/9/13
We’re repeating this procedure, due to that fact that days between a digestion and ligation shows to be ineffective. Hence, a ligation should be completed soon after digestion.
pSB1C3 | |
---|---|
Component | Amount (μL) |
EcoR1 | 2 |
Pst1 | 2 |
10x Buffer | 2 |
DNA | 2.5 |
PCR H2 | 11.5 |
Total | 20 |
Clostridial Origin Fragment | |
---|---|
Component | Amount (μL) |
EcoR1 | 2 |
Pst1 | 2 |
10x Buffer | 2 |
DNA | 10 |
PCR H2 | 4 |
Total | 20 |
Place in 37°C water bath for 15 minutes
Ran gel
Used wrong kit (Spin mini prep). Suppose to use PCR purification kit.
\Cleaned/purified digests
Clostridial Origin
pSB1C3
Ligation:
[(ng vector X size insert in Kb)/Size vector in Kb] X molar ratio of insert : vector = ng of insert
Since we used the wrong kit, we’re going to precipitate the DNA out (used digestion on May/9)
Phenol Chloroform Protocol pH 8:
Now we have 50μL of DNA
05/9/13
pSB1C3 = 2000 base pairs
Costridial Origin = 700 base pairs
Want to digest 500ng of backbone and 1:1 ratio of backbone to insert
pSB1C3 linearized backbone = 202.1 ng/μL
500ng of pSB1C3:
1:1 mole ratio of backbone to insert
175ng of insert:
pSB1C3 | |
---|---|
Component | Amount (μL) |
EcoR1 | 2 |
Pst1 | 2 |
10x Buffer | 2 |
DNA | 2.5 |
PCR H2 | 11.5 |
Total | 20 |
Clostridial Origin Fragment | |
---|---|
Component | Amount (μL) |
EcoR1 | 2 |
Pst1 | 2 |
10x Buffer | 2 |
DNA | 10 |
PCR H2 | 4 |
Total | 20 |
Place in 37°C water bath for 15 minutes
Back to top
05/9/13
Used QIAquick PCR Cleanup Kit and processed pSB1K3 and pSB1A3 linearized plasmid backbone PCR products
Nanodrop Quantification
Back to top05/9/13
Well Number | Sample |
---|---|
1 | Ladder |
2 | pSB1K3 (blue Hp) (fail: pipette tip fell into well) |
3 | pSB1K3 |
4 | pSB1A3 (blue Hp) |
5 | pSB1A3 |
05/8/13
Dilute plasmid backbones to 10ng/μL
Two reactions for pSB1K3 and pSB1A3 | |
---|---|
Component | Amount (μL) |
PCR High Fidelity Supermix | 97 |
Primer 1: SB prep 3P-1 | 1 |
Primer 2: SB prep 2Ea | 1 |
Template | 10ng |
pSB1K3
pSB1A3
Thermo Cycler (x38)
02/12/13
stuff stuff stuff stuff
Back to top05/1/13
To have eight reaction vials with 50μL supermix each. Extra is made for potential pipetting error.
Component | Amount (μL) |
---|---|
Amplification Buffer 10x | 50 |
dNTP Mixture (10 mM) | 15 |
50 mM MgSO4 | 10 |
Pfx DNA Polymerase (Hf) (Spin down) | 10 |
dH2O | 415 |
Total (two different backbones) | 500 |
PCR reaction of linearized backbone in each tube | |
---|---|
Component | Amount (μL) | PCR Supermix HF | 50 |
SB prep 3P-1 | 0.4 |
SB prep 2Ea | 0.4 |
DNA | 5ng |
pSB1K3
pSB1A3
For our eight reactions, multiply all values (except supermix) above by 10 and add/mix into 250μL of supermix
Final Reaction Tube Composition | |
---|---|
Component | Amount (μL) |
PCR Supermix | 250 |
SB prep 3P-1 | 4 |
SB prep 2Ea | 4 |
pSB1K3 DNA | 50ng = 2μL |
pSB1A3 DNA | 50ng = 0.4μL |
Alliquot 55μL of supermix , primers, and template into each tube
To prepare dNTP mix
PCR for linearized backbone (actual)
Four reaction of 1 backbone | |
---|---|
Component | Amount (μL) |
10x Buffer | 25 |
Polymerase Hf (spin) | 5 |
Mg | 5 |
dNTP mix | 7.5 |
DI H2O | 203 |
Forward Primer | 2 |
Reverse Primer | 2 |
Template | 1 (may need to dilute) |
Total | 250 |
To dilute pSB1A3:(135.8 ng/μL)
1μL Template
4μL PCR dH2O
We didn’t get what we were trying to amplify
Back to top Back to top04/28/13
Preformed as on pg. 15 with the following modifactions
Repeat 30x
32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin
PCR product was digested using QIAquick PCR purification kit
Note: If solution turns pick, the pH needs to be adjusted with 5μL of sodium acetate
Back to top04/24/13
For 0.5L
OR
04/19/13
Repeat 30x
The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had
Back to top04/19/13
PCR Supermix High Fidelity | 9.6mL |
Primer SB-prep 2Eb | 65μL |
Primer SB-prep 3P-1 | 65μL |
DNA Template | 100ng |
pSB1K3
pSB1A3
pSB1C3
High fidelity aliquots of 100μL for PCR
PCR
PCR Cleanup: Use Quiagen
Back to top04/9/13
Row 1 | |
---|---|
Lane | Sample |
1 | 1 Kb Plus Ladder |
2 | pIKM1 + RSA1 digest |
3 | pIKM1 |
4 | pAN1 + RSA1 digest |
5 | pAN1 |
85 Volts for 1 hour
Back to top04/8/13
Because we may have accidently stimulated the plasmid and mislabeled them previously. Since, no digestion of the plasmid pIKM1 from last weeks experiment is consistent with a methylating plasmid, today we are digesting both plasmids and running a gel.
This should verify that either we mixed up our plasmids, or we received the wrong plasmid.
1) Plasmid preparation of pAN1 and pIKM1 via Qiagen QiaPrep Spin Miniprep Unit instructions
2) Nanodrop quantification of plasmid DNA
3) Digest of pIKM1 and pAN1 with RSA1
pAN1
pIKM1
Compnents | pAN1 | pIKM1 |
---|---|---|
DNA | 10μL | 2μL |
RSA1 | 2μL | 2μL |
10x Buffer | 2μL | 2μL |
PCR Water | 6μL | 14μL |
TOTAL | 20μL | 20μL |
Placed in 42°C water bath for 15 minutes
Back to top04/5/13
Row 1 | |
---|---|
Lane | Sample |
1 | 1 Kb Plus Ladder |
2 | pSB1C3 + RSA1 + Xba + Invitrogen Buffer |
3 | pSB1C3 + RSA1 + Xba + Fermentas Buffer |
4 | pSB1C3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer |
5 | pSB1A3 + RSA1 + Xba + Invitrogen Buffer |
6 | pSB1A3 + RSA1 + Xba + Fermentas Buffer |
7 | pSB1A3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer |
8 | PIKM1 + RSA1 |
9 | pSB1K3 + Hind1 + Xba |
10 | Negative Control |
11 | PIKM1 |
12 | pSB1K3 |
04/3/13
Since we couldn’t find information about the Fermentas buffer needed for Xba1 and Hind3, we are going to set up our experiment in such a way that tests to see if using only the buffer from Fermentas, using the buffer from Invitrogen, or using a 1:1 mixture of the two will allow our digestion to occur in reactions where enzymes from different companies are being used.
Tubes will be labeled with the plasmid name, which enzyme used, and which buffer was used. For example
We incubated digests in 42°C water bath for 15 minutes
Back to top04/1/13
PPIKM1
Biobrick
Use a gel of 2% agarose
Regarding a digest, you typically want to match the company that produced the enzyme you’re using to the same company for the buffer. If they’re not the same, look up components and concentrations.
Back to top03/28/13
This looks to be the same size no matter the temperature. We hypothesize that either our primers are laying down non-specifically or they are oriented in the wrong direction. If it turns out our primers are fine, then we may have a different organis, than expected.
Back to top03/27/13
Preformed as outline on page 15 with the following modifications
Column | Temperature (μC) |
---|---|
2 | 50.2μC |
3 | 50.8μC |
4 | 51.7μC |
5 | 52.8μC |
6 | 54.1μC |
7 | 55.4μC |
8 | 56.7μC |
9 | 57.9μC |
10 | 58.8μC |
11 | 59.5μC |
To set up temperature gradient on thermocycler
03/25/13
PCR analysis was completed March 13, 2013
From these gels, we can see a band between 250 and 500 bases, which isn't the size of our clostridial origin. Assuming PCR worked correctly, we should see a band of approximately 1200 base pairs. Since we ran multiple cells with the same result, we are hypothesizing that an error was in the PCR reaction. Therefore, we are going to repeat PCR before we move forward by adjusting the annealing temperature.
Back to top03/15/13
Nanodrop Protocol
03/13/13
Making and Preparing Agarose Gel
Running Gel of Post-Digested pSB1C3 and pSB1A3