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Team:BYU Provo/Notebook/Metabolism/mayjune
From 2014.igem.org
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<p>Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights.</p> | <p>Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights.</p> | ||
<h3>May 22, 2014</h3> | <h3>May 22, 2014</h3> | ||
| - | Did plasmid preps on overnights from colonies 4-7. | + | <p>Did plasmid preps on overnights from colonies 4-7.</p> |
| - | Nanodropped: | + | <p>Nanodropped:</p> |
| + | <ui> | ||
| + | <li>206.1 ng/ul 260/280: 1.89</li> | ||
| + | <li>215.4 ng/ul 260/280: 1.89</li> | ||
| + | <li>400.2 ng/ul 260/280:1.85</li> | ||
| + | <li>170.0 ng/ul 260/280: 1.84</li> | ||
| + | <p>Set up restriction digests on New Plasmid (pIG91+BlaGenepIG102), and promoter plasmids to build final construct with promoter and the bla gene together.</p> | ||
| + | RD 5-221M, restriction digest of pIG102 cut with EcoR1 and Xba. <a href="http://www.igem.org">This is the iGEM link that eventually needs to be changed to *Restriction digest protocol</a> | ||
| - | + | <p>RD 5-222M, and RD 5-223M </p> | |
| - | + | <p>Same as above except: | |
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| - | RD 5-222M, and RD 5-223M | + | |
| - | Same | + | |
Enzymes used are EcoR1 HF and Spe1 HF | Enzymes used are EcoR1 HF and Spe1 HF | ||
Buffer 4 Used | Buffer 4 Used | ||
Two reactions, one containing promoter plasmid J23104 (5-222M), and the other J23111 (5-223M) | Two reactions, one containing promoter plasmid J23104 (5-222M), and the other J23111 (5-223M) | ||
| - | Ran Low Melt Gel at 90V for 46 min. | + | Ran Low Melt Gel at 90V for 46 min. </p> |
| - | Each of the Plasmids showed up, which was good for pIG102, where we want that to be our vector. Other RD only saw the plasmids and not the promoter part which we are trying to insert into pIG102…. | + | <p>Each of the Plasmids showed up, which was good for pIG102, where we want that to be our vector. Other RD only saw the plasmids and not the promoter part which we are trying to insert into pIG102….</p> |
| - | + | <h2>Week of May 31st</h2> | |
| - | + | <h3>May 27. 2014</h3> | |
Set up RD of promoter plasmids J23101, and J23106 | Set up RD of promoter plasmids J23101, and J23106 | ||
Used 3 RD enzymes because we will not be able to run on low melt and get out promoter part, so we want to ruin the plasmid the promoter is in so that it doesn’t relegate. Used Enzymes EcoRI, PstI, SpeI | Used 3 RD enzymes because we will not be able to run on low melt and get out promoter part, so we want to ruin the plasmid the promoter is in so that it doesn’t relegate. Used Enzymes EcoRI, PstI, SpeI | ||
Revision as of 00:00, 10 July 2014
BYU 2014 Notebook |
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May 6, 2014
Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.
May 8, 2014
Set up PCR to amplify Bla gene. This is the iGEM link that eventually needs to be changed to *Q5 protocol link Used psB1A3 plasmid as template with primers BI374 and BI375.
May 9, 2014
Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using This is the iGEM link that eventually needs to be changed to *GenElute Protocol Kit linkRan restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)
May 10, 2014
Restriction digests were run on the following samples: Promoter part plasmids BBa_J23102, and BBa_J23118, and Bla gene PCR product. SpeI HF, and XbaI were used with NEB buffer 4 in this protocol for each of the three samples. This is the iGEM link that eventually needs to be changed to *Restriction digest protocol
Week of May 17th
May 13, 2014
Figured out that we didn’t want to use the promoter vectors that I digested. We tried using pIG91 from the freezer, but the RD gel did not show any DNA present. Also did This is the iGEM link that eventually needs to be changed to *Sigma-Aldrich plasmid prep protocol according to the Sigma-Aldrich kit, for new pIG91.
May 15, 2014
Assisted in setting up new RD of pIG91.
Assisted Nano-dropping the recently purified pIG91 plasmisds, all concentrations were approximately 100ng/ul.
Assisted in setting up ligation of digested pIG91 (treated with CIP), with the digested Bla. Both were digested using SpeI HF and XbaI. This is so that we can then have our part in the iGEM registry plasmid to allow for quick and easy future cloning.
Week of May 24th
May 20, 2014
Set up colony PCR. Chose 8 colonies from the transformation and streaked colonies onto another plate. Colonies 1-7 were white colonies. We chose colony 8 as a red colony to act as a negative control. Made a master mix (10x) the Taq PCR ProtocolThis is the iGEM link that eventually needs to be changed to *Taq PCR protocol
May 21, 2014
Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights.
May 22, 2014
Did plasmid preps on overnights from colonies 4-7.
Nanodropped:
Set up restriction digests on New Plasmid (pIG91+BlaGenepIG102), and promoter plasmids to build final construct with promoter and the bla gene together.
RD 5-221M, restriction digest of pIG102 cut with EcoR1 and Xba. This is the iGEM link that eventually needs to be changed to *Restriction digest protocolRD 5-222M, and RD 5-223M
Same as above except: Enzymes used are EcoR1 HF and Spe1 HF Buffer 4 Used Two reactions, one containing promoter plasmid J23104 (5-222M), and the other J23111 (5-223M) Ran Low Melt Gel at 90V for 46 min.
Each of the Plasmids showed up, which was good for pIG102, where we want that to be our vector. Other RD only saw the plasmids and not the promoter part which we are trying to insert into pIG102….
