Team:Columbia-Cooper/GenspaceNotebook

From 2011.igem.org

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<div style="text-align:left">
<div style="text-align:left">
 +
<h2>July 5</h2>
 +
<p>- Grew out BBa_k325219 and BBa_k325909 in media C.</p>
 +
<p>- Restriction digest of BBa_k231000 and BBa_k231001 with EcoRI and PstI</p>
 +
<h2>July 15</h2>
 +
<p>- CDS7 isolation and ligation</p>
 +
 +
<h2>July 19</h2>
 +
<p>- NcoI and BamHI digest of pET Vector</p>
 +
 +
<h2>July 20</h2>
 +
<p>- Another attempt at NcoI and BamHI digest of pET Vector</p>
 +
 +
<h2>July 21</h2>
 +
<p>- Gel Purified pET Vector Digest </p>
 +
<p>- Grew out more pET vector</p>
 +
 +
<h2>July 24</h2>
 +
<p>- Received new NcoI and BamHI enzymes </p>
 +
<p>- pET Vector digest with new NcoI and new BamHI</p>
 +
<p>- pET Vector digest with old NcoI and old BamHI</p>
 +
<p>- pET Vector digest with new NcoI and another with old NcoI</p>
 +
<p>- pET Vector digest with BamHI and anther with old BamHI</p>
 +
 +
<h2>July 25</h2>
 +
<p>- Ran a gel with all pET Vector digests from July 24th</p>
 +
<p>- Fermentas transformation on pET Vector</p>
 +
 +
<h2>July 28</h2>
 +
<p>- CDS7 digest with NcoI and BamHI</p>
 +
<p>- Prepared plates of kanamycin and pET Vector</p>
 +
<p>- Oligo annealed with Silver Lab protocol</p>
 +
<p>- Maxiprep using ~50mL CDS7 growth</p>
 +
<p>- pET Vector inoculation</p>
 +
 +
<h2>July 29</h2>
 +
<p>- pET Vector digest with BamHI and NcoI</p>
 +
<p>- Gel purification of pET Vector digest</p>
 +
<p>- Inoculated more pET Vector</p>
 +
<p>- Inoculated C-media for transformations</p>
 +
<p>- Miniprep of pET Vector  </p>
 +
<p>- Ligated 4 oligos</p>
 +
<p>- Prepare transformation ready cells using C-media culture prepared the previous day</p>
 +
 +
 +
<h2>July 30</h2>
 +
<p>- Miniprep of pET Vector</p>
 +
<p>- Digestion of pET Vector with NcoI and BamHI</p>
 +
<p>- Oligo PCR</p>
 +
<p>-Digest pUC19 with EcoRI and PstI</p>
 +
 +
<h2>August 1</h2>
 +
<p>- Ran gels to verify pET and PUC double digests</p>
 +
<p>- Restriction digest of annealed oligo sequences with EcoRI and PstI</p>
 +
<p>- Oligo PCR verification</p>
 +
<p>- Digested pSB1C3 with EcoRI and PstI</p>
 +
 +
<h2>August 2</h2>
 +
<p>- pUC19 and pET Vector were isolated and gel purified </p>
 +
<p>- Miniprep of pUC19, pET Vector, and pSB1C3</p>
 +
 +
<h2>August 3</h2>
 +
<p>- Ligated pUC19 and all the oligos</p>
 +
 +
<h2>August 5</h2>
 +
<p>- PCR with new CDS7 was successful. Sung has “never seen a brighter 70 bp lane in [his] life”</p>
 +
<p>- pUC19 – quantum dot synthesis oligos worked</p>
 +
<p>- Miniprep, digest with EcoRI and PstI, and gel run of CDS7</p>
 +
 +
<h2>August 6</h2>
 +
<p>- Gel extraction of pUC19 and pET Vector compatible CDS7 amplicon</p>
 +
 +
<h2>August 7</h2>
 +
<p>- Inoculated oligo ligation cells in ampicillin media</p>
 +
<p>- Digested and ran more of oligo ligations</p>
 +
<p>- Inoculated more of the ligations </p>
 +
<p>- Inoculated 6 CDS7 colonies</p>
 +
 +
<h2>August 8</h2>
 +
<p>- Optimized PCR</p>
 +
 +
<h2>August 22</h2>
 +
<p>- Oligo annealed </p>
 +
<p>- Ethanol precipitation of DNA</p>
 +
 +
<h2>August 23</h2>
 +
<p>- Precipitation of CDS7 and restriction digest with EcoRI and PstI</p>
 +
<p>- Oligo annealed</p>
 +
 +
 +
<h2>August 24</h2>
 +
<p>- Dephosphorylated  backbone and created ligation mixtures</p>
 +
<p>- Annealed biobrick part</p>
 +
<p>- Made master mix for biobrick backbone</p>
 +
<p>- Oligo annealed</p>
 +
<p>- Digested pUC19 with EcoR1 and PstI</p>
 +
 +
<h2>August 30</h2>
 +
<p>- Attempted ligation of biobrick</p>
 +
 +
<h2>August 31</h2>
 +
<p>- Tested previous day’s ligation in a gel and it failed</p>
 +
 +
<h2>September 1</h2>
 +
<p>- pUC19 digest with EcoRI and PstI </p>
 +
<p>- Gel purification of pUC19 digest</p>
 +
<p>- Alcohol precipitated CDS7</p>
 +
 +
<h2>September 2</h2>
 +
<p>- Received new primers</p>
 +
<p>- Ran three PCR reactions, CDS7 forward with RBS, reverse, and control</p>
 +
<p>- Ran 1/100 dilution of gel</p>
 +
 +
<h2>September 7</h2>
 +
<p>- Another CDS7 PCR</p>
 +
 +
<h2>September 8</h2>
 +
<p>- Restriction digest of J140 with EcoRI and PstI</p>
 +
 +
<h2>September 9</h2>
 +
<p>- Ran gel of pET Vector, j140, pSB1C3, CDS7, and CDS7S</p>
 +
 +
<h2>September 13</h2>
 +
<p>- Restriction digest of master mix with EcoR1 and Pst1</p>
 +
<p>- Made a ligation mix</p>
 +
<p>- Transformed with ligation mix</p>
 +
 +
<h2>September 14</h2>
 +
<p>-  pET Vector digest with Nco1 and BamH1</p>
 +
<p>- CDS7 PCR</p>
 +
<p>- CDS7 digest with EcoRI and PstI</p>
 +
<p>- CDS7 digest with XbaI and PstI</p>
 +
<p>- Gel verification and purification of pET Vector and CDS7</p>
 +
<p>- Ligation of CDS7 EcoRI and PstI cut with pSB1C3</p>
 +
 +
 +
<h2>September 20</h2>
 +
<p>- VF and VR PCR verification</p>
 +
<p>- Restriction digest on pSB1C3 with Pst1 and EcoR1</p>
 +
<p>- Gel verification of digest</p>
 +
 +
<h2>September 21</h2>
 +
<p>- Verified base pair numbers of primers and verified they work</p>
 +
<p>- PCR of CDS7</p>
 +
<p>- Gel purification of CDS7</p>
 +
<p>- Digest CDS7 with EcoR1 and Pst1</p>
 +
<p>- Phosphotased pSB1C3</p>
 +
<p>- Made ligation mix with all the parts</p>
 +
<p>- Transformed with the ligation mix</p>
 +
 +
<h2>September 23</h2>
 +
<p>- Ligation mix from previous day failed</p>
 +
<p>- Re-did everything from September 21st except this time made a new master mix with different PCR settings</p>
 +
<p>- Ligated and transformed with the new master mix</p>
 +
 +
<h2>September 24</h2>
 +
<p>- Grew colonies overnight from previous days ligation and transformation</p>
 +
<p>- Grew out colonies with LB chloramphenicol </p>
 +
<p>- Made minipreps of CDS7</p>
 +
 +
<h2>September 26</h2>
 +
<p>- PCR a sequencing segment from plasmid with VF2 and VR</p>
 +
<p>- Sent segment off for sequencing</p>
 +
<p>- Reviewed other transformations</p>
</div>
</div>
</body>
</body>
</html>
</html>

Revision as of 00:34, 29 September 2011


Genspace Notebook

July 5

- Grew out BBa_k325219 and BBa_k325909 in media C.

- Restriction digest of BBa_k231000 and BBa_k231001 with EcoRI and PstI

July 15

- CDS7 isolation and ligation

July 19

- NcoI and BamHI digest of pET Vector

July 20

- Another attempt at NcoI and BamHI digest of pET Vector

July 21

- Gel Purified pET Vector Digest

- Grew out more pET vector

July 24

- Received new NcoI and BamHI enzymes

- pET Vector digest with new NcoI and new BamHI

- pET Vector digest with old NcoI and old BamHI

- pET Vector digest with new NcoI and another with old NcoI

- pET Vector digest with BamHI and anther with old BamHI

July 25

- Ran a gel with all pET Vector digests from July 24th

- Fermentas transformation on pET Vector

July 28

- CDS7 digest with NcoI and BamHI

- Prepared plates of kanamycin and pET Vector

- Oligo annealed with Silver Lab protocol

- Maxiprep using ~50mL CDS7 growth

- pET Vector inoculation

July 29

- pET Vector digest with BamHI and NcoI

- Gel purification of pET Vector digest

- Inoculated more pET Vector

- Inoculated C-media for transformations

- Miniprep of pET Vector

- Ligated 4 oligos

- Prepare transformation ready cells using C-media culture prepared the previous day

July 30

- Miniprep of pET Vector

- Digestion of pET Vector with NcoI and BamHI

- Oligo PCR

-Digest pUC19 with EcoRI and PstI

August 1

- Ran gels to verify pET and PUC double digests

- Restriction digest of annealed oligo sequences with EcoRI and PstI

- Oligo PCR verification

- Digested pSB1C3 with EcoRI and PstI

August 2

- pUC19 and pET Vector were isolated and gel purified

- Miniprep of pUC19, pET Vector, and pSB1C3

August 3

- Ligated pUC19 and all the oligos

August 5

- PCR with new CDS7 was successful. Sung has “never seen a brighter 70 bp lane in [his] life”

- pUC19 – quantum dot synthesis oligos worked

- Miniprep, digest with EcoRI and PstI, and gel run of CDS7

August 6

- Gel extraction of pUC19 and pET Vector compatible CDS7 amplicon

August 7

- Inoculated oligo ligation cells in ampicillin media

- Digested and ran more of oligo ligations

- Inoculated more of the ligations

- Inoculated 6 CDS7 colonies

August 8

- Optimized PCR

August 22

- Oligo annealed

- Ethanol precipitation of DNA

August 23

- Precipitation of CDS7 and restriction digest with EcoRI and PstI

- Oligo annealed

August 24

- Dephosphorylated backbone and created ligation mixtures

- Annealed biobrick part

- Made master mix for biobrick backbone

- Oligo annealed

- Digested pUC19 with EcoR1 and PstI

August 30

- Attempted ligation of biobrick

August 31

- Tested previous day’s ligation in a gel and it failed

September 1

- pUC19 digest with EcoRI and PstI

- Gel purification of pUC19 digest

- Alcohol precipitated CDS7

September 2

- Received new primers

- Ran three PCR reactions, CDS7 forward with RBS, reverse, and control

- Ran 1/100 dilution of gel

September 7

- Another CDS7 PCR

September 8

- Restriction digest of J140 with EcoRI and PstI

September 9

- Ran gel of pET Vector, j140, pSB1C3, CDS7, and CDS7S

September 13

- Restriction digest of master mix with EcoR1 and Pst1

- Made a ligation mix

- Transformed with ligation mix

September 14

- pET Vector digest with Nco1 and BamH1

- CDS7 PCR

- CDS7 digest with EcoRI and PstI

- CDS7 digest with XbaI and PstI

- Gel verification and purification of pET Vector and CDS7

- Ligation of CDS7 EcoRI and PstI cut with pSB1C3

September 20

- VF and VR PCR verification

- Restriction digest on pSB1C3 with Pst1 and EcoR1

- Gel verification of digest

September 21

- Verified base pair numbers of primers and verified they work

- PCR of CDS7

- Gel purification of CDS7

- Digest CDS7 with EcoR1 and Pst1

- Phosphotased pSB1C3

- Made ligation mix with all the parts

- Transformed with the ligation mix

September 23

- Ligation mix from previous day failed

- Re-did everything from September 21st except this time made a new master mix with different PCR settings

- Ligated and transformed with the new master mix

September 24

- Grew colonies overnight from previous days ligation and transformation

- Grew out colonies with LB chloramphenicol

- Made minipreps of CDS7

September 26

- PCR a sequencing segment from plasmid with VF2 and VR

- Sent segment off for sequencing

- Reviewed other transformations