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Team:BYU Provo/Notebook/Auxotrophy/julyaug
From 2014.igem.org
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</table> | </table> | ||
| - | <h2 style="003468">Week of July | + | <h2 style="003468">Week of July 11</h2> |
| + | <h3>July 7</h3> | ||
| + | <p> (TR,CB) We began the cloning process over again with recreating the front and back halves of our insert via PCR using the Phusion polymerase.</p> | ||
| + | <h3>July 8</h3> | ||
| + | <p> (TR) Ran our PCR product out on a gel and found the PCR was unsuccessful yesterday. Will redo tomorrow.</p> | ||
| + | <h3>July 9</h3> | ||
| + | <p> (TR,CB) Again performed PCR to make our front and back halves, this time using q5 as our DNA polymerase. Running it on a gel afterward, we found we were successful. There was a band in both lanes at about the 500bp mark, just what we were looking for.</p> | ||
| + | |||
| + | <h2>Week of July 18</h2> | ||
| + | <h3>July 14</p> | ||
| + | |||
<h2>Week of August 15</h2> | <h2>Week of August 15</h2> | ||
<h3>Aug 11</h3> | <h3>Aug 11</h3> | ||
| - | <p> (TR,CB) | + | <p> (TR,CB) The decision was made that, since we are probably not going to use <i>N multiformis</i> as our chassis any more, we will just focus on <i>N eutropha</i> for now.</p> |
<h3>Aug 13</h3> | <h3>Aug 13</h3> | ||
<p> (TR) I went ahead and designed primers to be used with <i>N eutropha</i> to knock out the SerB gene and hopefully make it auxotrophic for serine. Here they are;</p> | <p> (TR) I went ahead and designed primers to be used with <i>N eutropha</i> to knock out the SerB gene and hopefully make it auxotrophic for serine. Here they are;</p> | ||
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<h2>Week of August 22</h2> | <h2>Week of August 22</h2> | ||
<h3>Aug 18</h3> | <h3>Aug 18</h3> | ||
| - | <p> (TR,CB) We | + | <p> (TR,CB) We received the primers for our <i>N eutropha</i> auxotrophy insert, but are unable to start PCR on it because we have no template. We have decided that in the mean time, we are going to try and finish our cloning for <i>N multiformis</i>. We took template for <i>N multiformis</i> genomic DNA from a frozen stock to synthesize the 500bp front and back halves of our insert again. Then we used the Common Procedure for q5 high fidelity DNA Polymerase to do PCR.</p> |
<h3>Aug 20</h3> | <h3>Aug 20</h3> | ||
<p> (TR,CB) Ran a gel electrophoresis on Monday's PCR products. We saw that both, the front and back halves of our insert were present around the 500bp mark.</p> | <p> (TR,CB) Ran a gel electrophoresis on Monday's PCR products. We saw that both, the front and back halves of our insert were present around the 500bp mark.</p> | ||
Revision as of 02:36, 3 October 2014
BYU 2014 Notebook |
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Week of July 11
July 7
(TR,CB) We began the cloning process over again with recreating the front and back halves of our insert via PCR using the Phusion polymerase.
July 8
(TR) Ran our PCR product out on a gel and found the PCR was unsuccessful yesterday. Will redo tomorrow.
July 9
(TR,CB) Again performed PCR to make our front and back halves, this time using q5 as our DNA polymerase. Running it on a gel afterward, we found we were successful. There was a band in both lanes at about the 500bp mark, just what we were looking for.
Week of July 18
July 14
Week of August 15
Aug 11
(TR,CB) The decision was made that, since we are probably not going to use N multiformis as our chassis any more, we will just focus on N eutropha for now.
Aug 13
(TR) I went ahead and designed primers to be used with N eutropha to knock out the SerB gene and hopefully make it auxotrophic for serine. Here they are;
Forward 1: overhang compatibility - bamHI - front of front 500 bp homology block
GAT - GGATCC - CGATACCTATGGGCATATTGCAG
Reverse 1: Reverse complement of ‘Forward 2’ primer (SOEing primer 2)
TCGAGACCGACGTGATTAAG - TGTATATCTGTACCTTGAAT
Forward 2: front and back of serB coding strand (SOEing primer 1)
ATTCAAGGTACAGATATACA - CTTAATCACGTCGGTCTCGA
Reverse 2: overhang compatibility - speI - reverse complement of back 500 bp homology block
ATC - ACTAGT - AGGAACGCCCCGTGATTATTGCG
Week of August 22
Aug 18
(TR,CB) We received the primers for our N eutropha auxotrophy insert, but are unable to start PCR on it because we have no template. We have decided that in the mean time, we are going to try and finish our cloning for N multiformis. We took template for N multiformis genomic DNA from a frozen stock to synthesize the 500bp front and back halves of our insert again. Then we used the Common Procedure for q5 high fidelity DNA Polymerase to do PCR.
Aug 20
(TR,CB) Ran a gel electrophoresis on Monday's PCR products. We saw that both, the front and back halves of our insert were present around the 500bp mark.
Week of August 29
Aug 25
(TR,CB) Performed SOEing PCR on the front and back halves of insert to go into pSR47s plasmid. We used the high fidelity q5 DNA polymerase.
Aug 26
(TR) Electrophoresed our SOEing PCR product. The gel came out with only a band around 500bp, so something didn't work correctly.
Aug 27
(TR,CB) Started another SOEing PCR to try and prepare the insert for cloning, using q5 DNA polymerase. Also started overnight cultures for some plasmid preps of the pSR47s plasmid, so that we have plenty of vector to work with.
Aug 28
(TR,CB) Performed a plasmid prep of the overnight cultures with pSR47s. Ran a gel electrophoresis of our PCR product and found that the SOEing didn't work again.
