Team:RHIT/Notebook.html

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                    <h4><a href="#mayw1">May</a></h4>
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                 <p>The systems (one for E. coli and one for Yeast) described during the project explanation are broken up into their parts below. Each of the parts combined create a system that has a specific function but the two constructs together create a positive feedback loop that allows the two organisms to stay in close proximity. </p>
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                 <p>The systems (one for <i><i>E. coli</i></i> and one for Yeast) described during the project explanation are broken up into their parts below. Each of the parts combined create a system that has a specific function but the two constructs together create a positive feedback loop that allows the two organisms to stay in close proximity. </p>
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        <div class = "title">May</div>
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06-25-13</br>
06-25-13</br>
Safety Training round 2! Calibrated pipettes.  Inoculated liquid cultures with pre-plated colonies.
Safety Training round 2! Calibrated pipettes.  Inoculated liquid cultures with pre-plated colonies.
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Concentrated DNA from Friday and Monday with Ethanol precipitation, ran gel for concentration, and Ligated fragments.  Transformed and plated competent E. Coli with ligation mixture.  Made L-Amp plates and L-Amp broth. </br>  
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Concentrated DNA from Friday and Monday with Ethanol precipitation, ran gel for concentration, and Ligated fragments.  Transformed and plated competent <i><i>E. coli</i></i> with ligation mixture.  Made L-Amp plates and L-Amp broth. </br>  
Members: BATSMD, BAT</br></br>
Members: BATSMD, BAT</br></br>
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07-16-13</br>
07-16-13</br>
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No transformants observed from yesterday’s ligation. Performed ligation on previously cut samples of pRS416 and the Blue construct from 7-11-13. The ligation was incubated at 16°C overnight instead of at room temperature for 1 hour as previously done. Created stock yeast cells: .750mL of 30% glycerol and .750mL of yeast YPD culture. The E. Coli cells plated yesterday grew on LB plate. These cells were then used to inoculate liquid LB culture. </br>
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No transformants observed from yesterday’s ligation. Performed ligation on previously cut samples of pRS416 and the Blue construct from 7-11-13. The ligation was incubated at 16°C overnight instead of at room temperature for 1 hour as previously done. Created stock yeast cells: .750mL of 30% glycerol and .750mL of yeast YPD culture. The <i><i>E. coli</i></i> cells plated yesterday grew on LB plate. These cells were then used to inoculate liquid LB culture. </br>
Members: BT</br></br>
Members: BT</br></br>
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Latest revision as of 08:54, 26 September 2013

Notebook

The systems (one for E. coli and one for Yeast) described during the project explanation are broken up into their parts below. Each of the parts combined create a system that has a specific function but the two constructs together create a positive feedback loop that allows the two organisms to stay in close proximity.

June
Week 2

06-12-13
Plated Blue Construct and pRS416 from -80 stock: two plates each. Added AMP to LB liquid broth. Members: BAT

06-13-13
Moved 2 colonies from each plate onto 5 ml of liquid media after allowing for 24 hours of growth.
Members: BAT

06-14-13
Ran minipreps of liquid cultures, gel using 1ul of prep ~100ng/ul. Digest 7ul of prep with .5 ul EcoRI/SpeI in 1X CutSmart and BSA. Ran Gel of Digest, extracted bands and used ethanol/NaCl precipitation to concentrate all samples of each type into one sample. Ran gel with 2ul of product, no bands. 15.5 hours
Members: BAT

June
Week 3
06-17-13
Received most reagents (sans Phenol-Chloroform), autoclaved microcentrifuge tubes and pipet tips, activated DEAE cellulose membranes, made stock solutions.
Members: BAT

06-21-13
Received phenol chloroform. Digested pRS416 and Blue construct, Extracted from gel using DEAE cellulose method.
Members: BAT

06-22-13
Ran gel to determine if extraction method worked. Had low concentrations of the blue construct and 416.
Members: AT

June
Week 4
06-24-13
Safety Training for everyone! Poured plates of YPD, CSM-His, and CSM-Ura. Digested 6ul of mini prep with .5 ul SpeI/EcoRI in a 10 ul reaction for 3 hours. Inactivated at 80° C for 1 hour. Extracted from gel using DEAE cellulose membranes. Stored in -20° C. Streaked 2 plates of the Blue construct and pRS413 each.
Members: RBATSMD, AT

06-25-13
Safety Training round 2! Calibrated pipettes. Inoculated liquid cultures with pre-plated colonies. Concentrated DNA from Friday and Monday with Ethanol precipitation, ran gel for concentration, and Ligated fragments. Transformed and plated competent E. coli with ligation mixture. Made L-Amp plates and L-Amp broth.
Members: BATSMD, BAT

06-26-13
Safety Training round 3! Learned how to perform mini preps. Inoculated 40 liquid cultures with colonies on transformed plates.
Members: BATSMD, AT

06-27-13
Safety Training round 4! Learned how to run and read a gel and how to handle Ethidium Bromide. Performed mini preps on 27 of the liquid cultures. Ran a gel of the results. No colonies had an 8kb band. Possible problem involving ligase?
Members: BATSMD, BAT

July
Week 1
Waited to get parts.
July
Week 2
07-08-13
Streaked plates with colonies to restart process.
Members: BAT

07-09-13
Mini Preps of liquid cultures pRS416 and Blue construct followed by an agarose gel of the Mini Prep samples. Digested pRS416 and the Blue construct with EcoR1 and Spe. Ran an agarose gel of digested DNA. Performed DNA extraction of pRS416 and the Blue construct from agarose gel.
Members: BAT

07-10-13
Used Mini Prep kit to concentration DNA from extraction. Ran an agarose gel to see if the DNA extraction was successful. Unsuccessful extraction was decided. New liquid cultures of LB AMP were inoculated with pRS416 and the Blue construct.
Members: BT

07-11-13
Mini preps were performed on the new liquid cultures. Ran an agarose gel of the Mini Preps. Digested pRS416 and the Blue construct with Spe. for 1 hour then digested the same sample with EcoR1 for 3 hours. BSA was added to the digestion along with the enzyme and cut smart buffer. Ran an agarose gel of digested DNA. Began DNA extraction of pRS416 and the Blue construct from gel by running the DNA from the gel onto the membrane and storing the membranes in high salt buffer in the 4°C fridge. Received Yeast cell strain and plated on YPD.
Members: BT

07-12-13
Extracted DNA from membrane and completed protocol for the extraction of DNA from a gel from yesterday’s sample. Performed ligation and transformation of digested pRS416 and the Blue construct. Ran an agarose gel for samples of before and after the ligated DNA. The gel showed a failed ligation. Two ligations were set up: one using Dr. Anthony’s ligase and the other using Dr. Brandt’s ligase.
July
Week 3
07-15-13
Performed ligation and transformation using the same DNA from extraction on 7-11-13 and 7-12-13. Ran a gel of the before and after ligations from the day. Only the before ligation DNA for Dr. A’s sample could be observed on the gel. It appeared to be pRS416 digested DNA only in the sample. Received E.coli cell strain and plated it on LB agar. Made new LB agar plates. Inoculated YPD culture with yeast cell strain colony from YPD agar plated 7-11-13.
Members: BT

07-16-13
No transformants observed from yesterday’s ligation. Performed ligation on previously cut samples of pRS416 and the Blue construct from 7-11-13. The ligation was incubated at 16°C overnight instead of at room temperature for 1 hour as previously done. Created stock yeast cells: .750mL of 30% glycerol and .750mL of yeast YPD culture. The E. coli cells plated yesterday grew on LB plate. These cells were then used to inoculate liquid LB culture.
Members: BT

07-17-13
Ran an agarose gel of before and after ligation samples to observe the success of the ligation from 7-16-13. Transformed ligated DNA. Started again from uncut fragment DNA of pRS416 and the Blue construct with individual digests this time not using BSA. Digested with Spe for 1 hour then EcoR1 for 3 hours.
Members: BT

07-18-13
No Transformants observed from 7-17-13 transformation. Ran a gel of yesterday’s digestion and extraction the DNA from the gel.
Members: BT

07-19-13
Ran a gel to confirm the DNA extraction was successful. From this gel the relative concentration of 1μL of the extracted DNA of pRS416 and the Blue construct was determined. From this concentration the amount of DNA needed for the ligation of pRS416 and the Blue construct was determined. A ligation was set up. 1μL of DNA for before ligation sample was obtained for the gel. The before ligation sample and the ligation sample were allowed to incubate at 16°C overnight.
Members: BT

07-20-13
1μL for after ligation sample was obtained for gel. Transformed ligated DNA onto LB AMP plates. Ran a gel showing the before and after ligation. The gel shows a somewhat successful ligation.
Members: BT

July
Week 4/5
07-22-13
No colonies were seen from 7-20-13 transformation. Transformation sample from 7-20-13 was spun down and resuspended in 200μL of LB liquid. This was plated on 2 LB plates. Four transformations of ligation mix using 5μL instead of 1μL of sample from 7-19-13 were performed.
Members: BT

07-23-13
No colonies were seen. The transformation sample from 7-22-13 was spun down and the cells were resuspended in 200μL. 100μL of three of these samples were plated on 6 LB AMP plates and 200μL of one these samples was plated on one plate. The plates were allowed to grow overnight at 37°C.
Members: BT

07-24-13
No colonies were seen from the transformation. We began the process back from the DNA from the Mini Preps on 7-11-13. The samples of pRS416 and the Blue construct were digested with Spe then EcoR1 as performed on 7-17-13.
Members: BT

07-25-13
Ligated blue construct and pRS416 overnight.
Members: BAT

07-26-13
Performed transformations with the ligation mixtures, received yeast construct in mail and performed transformation with it as well.
Members: BAT

07-27-13
Picked colonies from yeast construct and ligation plates, started liquid cultures.
Members: AT

07-28-13
Inoculated 5 ml cultures from 1 ml cultures.
Members: AT

07-29-13
Ran minipreps of colonies, performed a diagnostic digest with EcoRI and XbaI. Ran digest on gel, results were unclear and will be repeated tomorrow.
Members: BAT

07-30-13
Digested minipreps and ran on a gel, yeast construct size suggested it was the desired size, XbaI cut of blue construct revealed it was not the correct transformant. Consolidated minipreps of blue construct, pRS416, and the yeast construct, performed sequential digest with EcoRI and SpeI.
Members: BAT

07-31-13
Ran gel of sequential digests, extracted fragments from gel. Ran second gel to determine concentration of fragments.
Members: AT

August
Week 1
08-01-13
Ligated extracted fragments overnight at 16° C.
Members: AT

08-02-13
Transformed cells with ligation mix.
Members: AT

08-03-13
No transformed colonies were found on the plates.
Members: A

August
Week 2
08-05-13
Made more L-AMP media and L-CHL media. Started ligation of Blue construct with pRS416 and pRS416 and the Yeast Construct.
Members: AT

08-06-13
Transformed cells with ligation mixtures.
Members: AT

08-07-13
No cells found on plates. Digested Yeast Construct and pSB1C3 with EcoR1 and Pst1.
Members: AT

08-08-13
Ran gel of yeast construct. Isolated fragment from gel. Ran gel of the isolated fragment.
Members: AT

08-09-13
Ligated yeast construct and pSB1C3 overnight.
Members: AT

August
Week 3
08-12-13
Transformed cells with ligation mixture.
Members: AT

08-13-13
No plates had colonies.
Members: AT

08-14-13
Ran transformation of ligation mix. Cleaned up lab.
Members: AT

08-15-13
No transformants found on plates. Cleaned up lab.
Members: AT

08-16-13
Cleaned up lab, final storage of materials.
Members: AT


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