Team:Harvard/Template:NotebookDataJuly3

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(Transformation for OZ plasmids)
 
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====PCR on minipreps to check cross junction====
====PCR on minipreps to check cross junction====
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PCR was done on the miniprep products to check for the cross junction length, expected band size of ~1.4 kb.  The protocol used was the same as [[#PCR of expression plasmid cross-junction|the previous time]].  In addition to PCR-ing the miniprep products, a positive control was also included, which was the finished Zif268 + omega + spec backbone plasmid (this was the first successful junction PCR we obtained, and so it should work again).
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PCR was done on the miniprep products to check for the cross junction length, expected band size of ~1.4 kb.  The protocol used was the same as the previous time.  In addition to PCR-ing the miniprep products, a positive control was also included, which was the finished Zif268 + omega + spec backbone plasmid (this was the first successful junction PCR we obtained, and so it should work again).
====Replating final samples for 78, 79, 82====
====Replating final samples for 78, 79, 82====
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===Team Wolfe===
===Team Wolfe===
'''SNOWMAN!'''
'''SNOWMAN!'''
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[[File: snowman1.jpeg|left|Our snowman Persikov]]
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[[File: HARVSnowman1.jpeg|left|Our snowman Persikov]]
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[[File: snowman3.jpeg|none|Brandon with his creation]]
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[[File: HARVSnowman3.jpeg|none|Brandon with his creation]]
'''EcNR2 lambda red (Noah's prep):'''
'''EcNR2 lambda red (Noah's prep):'''
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**Placed colonies in 20 μL of water and used 1 μl for a 20 μL PCR reaction
**Placed colonies in 20 μL of water and used 1 μl for a 20 μL PCR reaction
**Then took 10 μL from 20 μL dilution and added to 100 μL of LB in 96 well plate and put in 30°C fridge, at 3:19 pm
**Then took 10 μL from 20 μL dilution and added to 100 μL of LB in 96 well plate and put in 30°C fridge, at 3:19 pm
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**Same protocol as [[Protocols#PCR|KAPA PCR]] with the following changes made
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**Same protocol as [https://2011.igem.org/Team:Harvard/Protocols#PCR KAPA PCR] with the following changes made
***Step 3, gradient for annealing, 55°C - 77°C, for a range of 55,57,59,61 and 63°C for the two rows of 5 PCR tubes each (placed leftmost)
***Step 3, gradient for annealing, 55°C - 77°C, for a range of 55,57,59,61 and 63°C for the two rows of 5 PCR tubes each (placed leftmost)
***Step 4, Extension, 1 min
***Step 4, Extension, 1 min
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**Name: TOLCGRAD, PCR5 machine.</div>
**Name: TOLCGRAD, PCR5 machine.</div>
<div id="719" style="display:none">
<div id="719" style="display:none">
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==July 19th==
==July 19th==
===Team ZF===
===Team ZF===
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====Minipreps of colonies and controls====
====Minipreps of colonies and controls====
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Our minipreps from [[#Colony minipreps|Saturday]] had low yields, especially for culture 85. We suspect this is why the PCR [[#Gel analysis of miniprep junction PCR|on Sunday]] did not work for this sample. Unfortunately, 85.3 and 85.4 did not grow even after being left in the shaker overnight. These were two cultures we made by picking new colonies from plate 85. This may have occurred because we did not actually pick the colony, or the colony was a cheater. Thus, we performed minipreps on 80.1, 80.2, 81.1, 81.4 (the controls, chosen for their wide range of concentrations after our first miniprep), and 85.1 and 85.2.  
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Our minipreps from Saturday had low yields, especially for culture 85. We suspect this is why the PCR [[#Gel analysis of miniprep junction PCR on Sunday did not work for this sample. Unfortunately, 85.3 and 85.4 did not grow even after being left in the shaker overnight. These were two cultures we made by picking new colonies from plate 85. This may have occurred because we did not actually pick the colony, or the colony was a cheater. Thus, we performed minipreps on 80.1, 80.2, 81.1, 81.4 (the controls, chosen for their wide range of concentrations after our first miniprep), and 85.1 and 85.2.  
In order to figure our why our minipreps were resulting in unexpectedly low yields and increase our yields:
In order to figure our why our minipreps were resulting in unexpectedly low yields and increase our yields:
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====PCR of miniprep products====
====PCR of miniprep products====
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We then ran a PCR for the cross-junction on all 6 (80.1, 80.2, 81.1, 81.4, 85.1, 85.2) samples, using [[#PCR of expression plasmid cross-junction|our usual cross junction protocol]].  
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We then ran a PCR for the cross-junction on all 6 (80.1, 80.2, 81.1, 81.4, 85.1, 85.2) samples, using our usual cross junction protocol.  
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====Designing ultramers for Zif268, OZ052, and OZ123====
====Designing ultramers for Zif268, OZ052, and OZ123====
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In preparation for designing our 96-well practice plate, we designed ultramers for our three positive controls Zif268, OZ052, and OZ123.  These ultramers contain the Type II binding sites as well as the F2/F3 fingers.  Since the [[Lab_Notebook#Design_of_Plate_Practice_Sequences|practice plate]] will contain oligos that encode for F1, we can use these three positive controls to practice and ensure that we secure the technique of using the Type II binding sites to swap in F1 into our expression plasmids.  The ultramers have not yet been ordered.
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In preparation for designing our 96-well practice plate, we designed ultramers for our three positive controls Zif268, OZ052, and OZ123.  These ultramers contain the Type II binding sites as well as the F2/F3 fingers.  Since the practice plate will contain oligos that encode for F1, we can use these three positive controls to practice and ensure that we secure the technique of using the Type II binding sites to swap in F1 into our expression plasmids.  The ultramers have not yet been ordered.
===Team TolC===
===Team TolC===
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====Construction of OZ052 and OZ123: Joining the Finger with Homology regions====
====Construction of OZ052 and OZ123: Joining the Finger with Homology regions====
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Previously, we did a PCR to [[Lab_Notebook:_July#Overlap_PCR_of_OZ052_and_OZ123|reassemble the ultramers]] for OZ052 and OZ123, but they only coded strictly for F1/F2/F3 and did not have any overhang homology with any surrounding code.  Thus, we performed PCR to add these homology overhangs so that we can integrate them with the omega subunit as well as the spec backbone to create a final expression plasmid.
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Previously, we did a PCR to reassemble the ultramers for OZ052 and OZ123, but they only coded strictly for F1/F2/F3 and did not have any overhang homology with any surrounding code.  Thus, we performed PCR to add these homology overhangs so that we can integrate them with the omega subunit as well as the spec backbone to create a final expression plasmid.
The program we used was 55* annealing temperature, and a 15 second extension time.  Gel results pictured below.
The program we used was 55* annealing temperature, and a 15 second extension time.  Gel results pictured below.
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Our concentrations were better than they have been in the past; notably, after the addition of buffer P2, our solution actually turned a bright blue (indicating lysis of the cells), which we had not seen before.
Our concentrations were better than they have been in the past; notably, after the addition of buffer P2, our solution actually turned a bright blue (indicating lysis of the cells), which we had not seen before.
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We then ran a PCR on our miniprep product, to check for our desired insert. We used our [[#PCR of expression plasmid cross-junction|usual protocol]] for a PCR of the expression plasmid cross-junction.   
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We then ran a PCR on our miniprep product, to check for our desired insert. We used our usual protocol for a PCR of the expression plasmid cross-junction.   
{|
{|
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====Sending out our expression colonies for sequencing====
====Sending out our expression colonies for sequencing====
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We sent out the samples from [[#Expression Plasmid Sequencing Results|yesterday]] that had no more than 1 SNP for reverse sequencing. In addition, we also sent out 78 and 79 for forward and reverse sequencing.  
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We sent out the samples from yesterday that had no more than 1 SNP for reverse sequencing. In addition, we also sent out 78 and 79 for forward and reverse sequencing.  
*Reverse sequencing: 77.1, 77.3, 77.4, 78.1, 80.1, 80.2, 80.4, 81.1, 81.4, 82.1, 83.4, 84.1, 84.4, Zif268
*Reverse sequencing: 77.1, 77.3, 77.4, 78.1, 80.1, 80.2, 80.4, 81.1, 81.4, 82.1, 83.4, 84.1, 84.4, Zif268
*Forward and reverse sequencing: 781, 78.2, 78.3, 78.4, 79.1, 79.2, 79.3, 79.4
*Forward and reverse sequencing: 781, 78.2, 78.3, 78.4, 79.1, 79.2, 79.3, 79.4
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====Gel Extraction and Isothermal Assembly of OZ fingers+homology====
====Gel Extraction and Isothermal Assembly of OZ fingers+homology====
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We ran a gel extraction of our PCR product from [[#Construction of OZ052 and OZ123: Joining the Finger with Homology regions|yesterday]], so that we could use it for isothermal assembly later.  Strangely, we got bands that were larger than our largest theoretical product.
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We ran a gel extraction of our PCR product from yesterday, so that we could use it for isothermal assembly later.  Strangely, we got bands that were larger than our largest theoretical product.
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*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>
<div id="722" style="display:none">
<div id="722" style="display:none">
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==July 22nd==
==July 22nd==
===Team ZF===
===Team ZF===
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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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To this end, we redid the PCRs for everything in preparation for the resequencing, using the junction primers and the previous protocol.  The resulting gels can be observed below:
{|
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====Transformation for OZ plasmids====
====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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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 [https://2011.igem.org/Team:Harvard/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.
===Team Wolfe===
===Team Wolfe===

Latest revision as of 20:53, 4 August 2011