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| ==May 25== | | ==MAY NOTEBOOK== |
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| * Typed up the notes from the Workshop.
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| * Investigated the transformation of E.coli with a coding region for appetite regulation protein PYY.
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| * Created waves for "Iron Genes", the appetite regulation project, and the team to-do list.
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-25-10| May 25, 2010]] |
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| ==May 26==
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-26-10| May 26, 2010]] |
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| * Reviewed Patrick's genetic circuit for controlled cell population size.
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-27-10| May 27, 2010]] |
| * Reread Hasty's "Synchronized Genetic
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| * Compiled "Needed Materials and Equipment" List
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-28-10| May 28, 2010]] |
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| ==May 27==
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-29-10| May 29, 2010]] |
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| * Held meeting with advisers and four of the team members.
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-30-10| May 30, 2010]] |
| ** Discussed the Spring Workshop
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| ** Discussed team goals
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| ** Discussed long term goals for Citadel iGEM
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| ** Planned for training session.
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| * Mined OWW to create a library of relevant protocols.
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| * Established the protocol for May Training Session.
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| ==May 28==
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/05-31-10| May 31, 2010]] |
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| * Printed off library of relevant literature.
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| * Learned sterile technique.
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| ** See physical notebook for all of my observations.
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| * Inoculated a plate and a slant with ''M.luteus'' as practice for wetlab work next week.
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| * Prepared media.
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| ** Prepared LB media for making ''E.coli'' electrocompetent tomorrow.
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| ** Prepared Glycerol for the same procedure.
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| * Reorganized the "Needed Materials and Equipment" List.
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| * Prepared to complete training session by reading over all protocols.
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| ==May 29== | | ==JUNE NOTEBOOK== |
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| * Observed Adam's Miniprep Technique as a refresher.
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-01-10| June 01, 2010]] |
| * Prepared 10% glycerol.
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| * Prepared 600ml ddH2O.
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| * Observed plate and slant ''of M.luteus'' made on 05-28.
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| * Finished taking inventory.
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| * Prepared for Electrocompetence Protocol execution on 05-31.
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| ===Protocol for Electrocompetent Cells===
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-02-10| June 02, 2010]] |
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| ''Adapted from the Knight Lab version.<br>
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-03-10| June 03, 2010]] |
| Protocol by Austin Che and annotated by Reshma Shetty based on online research.''<br>
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| ==Materials==
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-04-10| June 04, 2010]] |
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| ===Equipment===
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-06-10| June 06, 2010]] |
| *-80°C freezer
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| *37°C incubator
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| *Refrigerated centrifuge that accepts 225 mL culture tubes
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| ===Chemicals and reagents===
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-21-10| June 21, 2010]] |
| *~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.
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| *~600 mL sterile deionized water chilled to 4°C
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| * 50 mL sterile 10% glycerol in deionized water chilled to 4°C
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| *Ice bucket and ice
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| *Dry ice, ethanol bath
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| ===Supplies===
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-22-10| June 22, 2010]] |
| *Many 1.5 mL plastic tubes chilled to -80 °C
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| *14 mL culture tube for starter culture
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| *2 L flask for culture
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| *225 mL plastic tubes for centrifugation
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| *Pipets
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| ==Procedure==
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-23-10| June 23, 2010]] |
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| #Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better.
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-24-10| June 24, 2010]] |
| #Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media
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| # Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.
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| # Fast cool the centrifuge with the correct rotor to 4°C
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| # Pour the culture into two 225 mL centrifuge tubes.
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| # Place the tubes on ice for 15 minutes. <br> This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br>
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| # Centrifuge for 10 mins at 2000g at 4°C
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| # Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
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| # Centrifuge for 15 mins at 2000g at 4°C
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| # Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
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| # Hold on ice for 30 minutes
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| # Centrifuge for 15 mins at 2000g at 4°C
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| # Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol. <br> This can be optionally transferred to a 50 mL conical tube.
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| # Hold on ice for 30 minutes
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| # Centrifuge for 15 mins at 1500g at 4°C
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| # Remove the supernatant and add 500 μl of 10% glycerol
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| # Resuspend the cells in a final volume of approximately 1 ml
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| # Aliquot 50 μL per tube (tubes on ice)
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| # Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation.
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| # Store at -80°C
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| ==Notes== | | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-25-10| June 25, 2010]] |
| *'''[[User:Rshetty|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-26-10| June 26, 2010]] |
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-27-10| June 27, 2010]] |
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-28-10| June 28, 2010]] |
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/06-29-10| June 29, 2010]] |
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| | ==JULY NOTEBOOK== |
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-06-10| July 06, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-09-10| July 09, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-14-10| July 14, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-15-10| July 15, 2010]]<br> |
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| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-16-10| July 16, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-19-10| July 19, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-21-10| July 21, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-27-10| July 27, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/07-28-10| July 28, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-05-10| August 5, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-06-10| August 6, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-07-10| August 7, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-08-10| August 8, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-09-10| August 9, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-10-10| August 10, 2010]]<br> |
| | [[iGEM:The_Citadel/Notebook/Notes_Brian/08-12-10| August 12, 2010]]<br> |
