Team:Yale/Our Project/Notebook/Week 7

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iGEM Yale

our project

lab notebook: week 7 (7/19-7/25)

Monday 7/19--Investigation of how pre-ligation processing affects ligation results

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Gel Purification of PCR product

Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL

Self-ligation of phsABC to test restriction enzymes

Took the single enzyme digests from 7/16 and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase. In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C

Dual-purpose gel

Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well. In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)
Based on gel it is clear that gel extraction is not causing loss of phsABC fragment. The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint. In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site.

Double digest of gel-purified phsABC

While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C. The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water.

AvaII digestions to check for inhibition

Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.
In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above.

Separation of phsABC double digestion

Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI. Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water.

This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook Extra content This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook

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 <li>
-Monday 7/19--
+Monday 7/19--Investigation of how pre-ligation processing affects ligation results
 </li>
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 <div style="display:none;" id="monday">
+<!------------- Monday ------------->
+<ul>
+<b>Gel Purification of PCR product </b> <br/>
+<li>Ran the phsABC PCR product on a 1.0% agarose gel at 90 V versus a 1 kb ladder to see how clean the result is--see very strong bands at 3.6 kb as expected. Excised the bright band and ran a standard vacuum manifold <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract " gel extraction protocol </a> to give three samples with DNA concentrations of 73.1, 43.3, and 45.8 ng/uL</li>
+<b>Self-ligation of phsABC to test restriction enzymes </b> <br/>
+<li> Took the single enzyme digests from <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_6"> 7/16 </a> and ran a self-ligation of each with 17 uL of DNA solution, 2 uL of ligase buffer, and 1 uL T4 ligase.  In each case ran trials both with iGEM ligase and buffer and with Grindley lab ligase & buffer. Let the reactions run for 20 minutes at room temperature before heat-killing with 20 minutes at 80˚C </li>
+<b> Dual-purpose gel </b> <br/>
+<li> Ran a gel to accomplish two things: 1.Determine whether the phsABC fragment was still present after gel extraction 2. Determine based on dimer formation of singly digested phsABC fragments whether the digestion/ligation is working well.  In a 1.0% agarose gel run at 90 V had the following lane assignments, left to right: Lane 1--1 kb ladder, Lane 2--circular 8 kb pSB74 plasmid, Lane 3--1 kb ladder, Lane 4--phsABC from gel extraction, Lane 5--self ligation of SpeI-digested phsABC (iGEM ligase), Lane 6--self ligation of SpeI-digested phsABC (Grindley lab ligase), Lane 7--self ligation of EcoRI-digested phsABC (iGEM ligase), Lane 8--self ligation of EcoRI-digested phsABC (Grindley lab ligase)</li>
+<li> Based on gel it is clear that gel extraction is not causing loss of phsABC fragment.  The self-ligation results are less straightforward, as a dimerized phsABC should run near 7.2 kb (as it does faintly in lane 5), but the other self ligations have products that are strangely long and also very faint.  In the future will fun ligations much longer and will also test for the presence of some sort of inhibition of digestion by digesting the the phsABC fragment at a central AvaII cut site. </li>
+<b>Double digest of gel-purified phsABC </b> <br/>
+<li> While dual-purpose gel was running, started three double digestion reactions of phsABC, which were let to run in the thermocycler for eight hours at 37˚C before a twenty minute heat-kill at 80˚C.  The contents of the reaction were 5 uL of EcoRI buffer, 0.5 uL of BSA, 13.7 uL phsABC (at 73.1 ng/uL for 1 ug total), 1.8 uL of EcoRI, 1.8 uL SpeI, and 27.2 uL of water. </li>
+<b>AvaII digestions to check for inhibition </b> <br/>
+<li> Digested 3.3 uL of phsABC from PCR clean-up protocol (about 330 ng) with 3.6 uL of AvaII, 5 uL of NEB buffer 4, and 38.1 uL of water, once again letting the reaction run eight hours at 37˚C before a twenty minute heat-kill at 80˚C.</li>
+<li> In a similar check, digested 4.6 uL of phsABC isolated by gel extraction (about 340 ng) and digested it with 3.6 uL AvaII, 5 uL NEB buffer 4, and 36.8, running the reaction at the same time and temperature as above. </li>
+<b>Separation of phsABC double digestion </b> <br/>
+<li>Run two single digests of the purer phsABC taken from gel extraction protocol, one with EcoRI and one with SpeI.
+Each has 5 uL of EcoRI buffer, 0.5 uL BSA, 3.6 uL of the relevant enzyme, 7.7 uL of phsABC solution (about 330 ng DNA), and 33.2 uL of water. </li>
+</ul>
+<i>This day's labwork is also recorded on pages 71-74 of the hard copy lab notebook </i>
 <!------------- Monday ------------->
 Extra content