Team:Harvard/Template:NotebookDataJuly3

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(Created page with "<div id="715" style="display:none"> ==July 15th== ===Team Wolfe=== '''Thio kan-ZFB-wp-hisura optimizing PCR results:''' *The gel stab PCR produced a product of the right size, bu...")
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*Repeated lambda red using the same procedure. Got a similar time constant and noticed that the water negative control had more cells than the culture containing the insert, but we went ahead and plated 1mL of each on kan plates.
*Repeated lambda red using the same procedure. Got a similar time constant and noticed that the water negative control had more cells than the culture containing the insert, but we went ahead and plated 1mL of each on kan plates.
*Also replated the 7/20 lambda red culture to see if we can get any good cells (1.5mL).</div>
*Also replated the 7/20 lambda red culture to see if we can get any good cells (1.5mL).</div>
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<div id="722" style="display:none">
 
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==July 22nd==
 
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===Team ZF===
 
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====Sequencing Results of Expression Plasmids====
 
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The good news is that we managed to get a perfect sequence for each of our plasmids.  The bad news is that we do not know which sequences are the matches, since the names are all scrambled for some reason (i.e. sample 78.x does not align with FH bottom like it should, but instead aligns perfectly with another plasmid sequence).  We will thus need to resend our samples for sequencing to be able to choose the correct plasmids that match.  Below is a summary of the scrambled data from the sequencing we sent in yesterday:
 
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{|
 
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| align="center" style="background:#f0f0f0;"|'''ID'''
 
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| align="center" style="background:#f0f0f0;"|'''Is a perfect match with'''
 
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| align="center" style="background:#f0f0f0;"|'''Notes'''
 
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|-
 
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| Myc 198 (81)||78.2, 78.3||
 
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|-
 
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| FH Top (77)||81.4, Myc 981||83.4 has 1 deletion
 
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|-
 
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| CB Bottom (80)||84.4||78.1 has 5 deletions; Zif has 1 deletion
 
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|-
 
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| FH bottom (78)||n/a||84.1 deletion at beginning of F3
 
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|-
 
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| CB Top (79)||79.3||
 
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|-
 
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| Myc 981 (82)||78.4||
 
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|-
 
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| Zif 268||79.4||
 
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|-
 
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| Pos Con 55 (83)||79.2||79.3 has deletion at beginning of F3
 
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|-
 
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| Pos Con 16||79.1||
 
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|}
 
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To this end, we redid the PCRs for everything in preparation for the resequencing, using the junction primers and  [[#PCR of expression plasmid cross-junction|the previous protocol]].  The resulting gels can be observed below:
 
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{|
 
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|[[File:2011.7.22_78_and_79_for_sequencing_annotated.png|thumb|E-gel showing bands for samples 78.1-78.4 and 79.1-79.4, expected size of 1.4 kb]]
 
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|}
 
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{|
 
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|[[File:2011.7.22_rest_of_pcr_for_sequencing_annotated.png|thumb|Remaining samples, expected size of 1.4 kb]]
 
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|}
 
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From these PCRs, all the reactions look like they produced bands of the right size, a good indicator that the isothermal assemblies worked.  Only sequencing will tell whether they are truly the products that we are looking for.
 
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We sent these samples out for sequencing, making sure that all the samples were in the appropriate tubes.
 
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====Transformation for OZ plasmids====
 
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Today, we took the results of the isothermal assembly from yesterday and did chemical transformation to introduce the plasmids into the Top 10 ChemComp bacteria, using [[Protocols#Cultures|our protocol for chemical transformations]]. They were left to recover for approximately 1.5 hours. We plated 2 dilutions, 10 ul and 50 ul, and left them overnight at 37* in the incubator.
 
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===Team Wolfe===
 
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*Yesterday's lambda red plate had several large colonies--but so did the negative control plate. We will go ahead and do a PCR of the 1529620 locus (as previously described) on 12 of the colonies in case one of them happens to have the insert. The colonies were also grown up simultaneously in LB+kan.
 
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**Results: all of the colonies were cheaters
 
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[[File: 2011.07.22.thiokan insert check1(labeled).ong|thumb|left|1529620 locus PCR 7/22/11]]
 
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[[File: 2011.07.22.thiokan insert check2(labeled).png|thumb|none|1529620 locus PCR part 2 7/22/11]]
 
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*In case these colonies do not have the insert, as we suspect, we will repeat lambda red with the thio kan-ZFB-wp-his3-ura3 insert
 
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**1mL culture for each condition, 210 ng DNA
 
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**time constant for thio insert sample was 5.0
 
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===Team TolC===
 
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====Gel for Jun's MAGE rpoZ, rowD====
 
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*The gel appeared to have bands, so we are running a second PCR to confirm it</div>
 
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<div id="723" style="display:none">
 
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==July 23rd==
 
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===Team Wolfe===
 
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*Lambda red results: no real colonies, only cheaters.</div>
 

Revision as of 00:01, 3 August 2011