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Calendar of Events

Protocols

Team:Michigan/Protocols

Notebook

5/13/2011

Kevin, Ben, Candy, Brian, Alison

⇒ Unpacked supplies from last year

⇒ Restocked lab and created inventory

⇒ Training protocols written

5/19/2011

Kevin, Alena, Ben, Namun

⇒ PCR machine is now operable. Basic programs have been written, but not all the functions of the machine are known. No protocol for using the PCR machine has been written.

⇒ Usage of the autoclave was determined. No protocol exists for autoclaving yet.

⇒ 25 plates were made using 10g LB agar stock, 250mL deionized water, and 250μL ampicillin from vials left over in the stupid white box in the freezer. Original protocol called for distilled water-abbreviation "dH2O" was misinterpreted to mean deionized. Not known if the plates are viable due to this error. House-made protocols will use the following abbreviations to avoid this confusion:

∴Distilled water: dH2O

∴Double distilled water (from the machine): ddH2O

∴Deionized water: DI water or diH2O

A protocol has been written for plate production. As it depends on the autoclave protocol, however, it cannot be considered complete.

⇒ Simple inventorying took place, with the locations of pipettes, ethanol, hardware, and the master gas shutoff(!) determined.

⇒ Inventorying will continue tomorrow, and a PCR reaction may be attempted. Protocols for the PCR purification kit (Miniprep) will be modified during this time, assuming the PCR reaction proceeds. No method currently exists to determine PCR outcome, as no gels or gel mix is available in the lab. Along these lines, it may be beneficial to find an ultraviolet spectrophotometer to run these tests.

5/20/2011

Amy, Ben, Candy

⇒ Cells were transformed with extra 173 pGLO vector in accordance with the QT protocol. This used stock competent cells found in the refrigerator. Protocol modifications will be made accordingly.

⇒ Access to room 3152 is crucial, and no means of entry currently exists. This will be imperative if we require ddH2O, an autoclave, LB stock, a normal scale, large glassware, or any number of other items.

⇒ Due to lack of access to 3152, protocols requiring sterile water cannot be tested. Sterile water exists in 3151A, but is 3 years past its expiration date.

⇒ No source of agarose or ethidium bromide has been found, so electrophoresis protocols cannot be tested. Interestingly, there exists plenty of loading dye.

⇒ Large amounts of stock compounds IE ethanol belong to the room and are not owned by us. Currently they are being used. This may cause problems, especially if we start using more expensive materials we still don't own.

On a side note, the contrast between the physical sign-in sheet in 3151 and the online form on ctools is rather confusing-it may be better to just use the online one, and have people log their hours individually.

5/22/2011

Ben ⇒ Two containers of 500mL LB broth were produced and autoclaved. 12.5g of LB powder was combined with 500mL water for each container.

5/24/2011

Ben ⇒ Six Erlenmeyer flasks of diH2O were autoclaved and are now sterile. Both sets of glassware reside on the shelf next to the incubator.

5/25/2011

Kevin, Alison, Ben, Chris

Had discussion with Dennis and Mark from the USB labs, learned some important points.

⇒ We aren't allowed to just "use" chemicals that are already in the USB that don't belong to us. I am working on ordering more of the basics right now. We are allowed to use the equipment in the room. However, if you don't know how to work a piece of equipment, please ask first!

⇒ Dennis and Mark are two very knowledgable supervisors who are conveniently generally very close to our lab room. If there is any equipment that you need assistance with, you can try to ask them. They are in an office close to our lab, I believe in room 3159. Just knock and be polite!

⇒ There are been some theft issues in the USB lately, so we need to be careful with keeping our door locked. Also, Dennis and Mark don't necessarily know our faces, so they won't necessarily know that we aren't actually trying to rob the USB. Additionally, our own Marc wants to know when we will be in the lab so if possible he could come in and help. If you are going to be in the lab unplanned, please email both Marc and CC Dennis.

⇒ A few more safety things when it comes to the lab- the outside door locks at night (I believe after 9). Make sure you bring your Mcard so you can get into the building after hours if that is needed. Also, the elevators in the USB change at night. One goes from P up to 4 and the other goes from P down to parking. Just keep this in mind. If you have your Mcard, you'll be able to get around the building.

⇒ A few other people that won't be expecting us if we are in the lab all night are the custodial staff, who will most likely call DPS on us. While this would make for a fantastic story, we don't want it happen. If there comes a time when we are in the lab past midnight, also let Dennis know at a decent hour so he can ensure we don't get DPS called on us.

Kevin and Alison tried to run a gel today, only to find that we lacked the necessary reagents and some equipment. We were able to borrow some equipment from Mark: microwave, transilluminator, and an autopipetter. Alison will buy some reagents tomorrow and we will try again.

5/26/2011

Kevin, Alison, Ben, Namun, Candy, David

⇒ Gel materials have been purchased by Alison the Great, including:

∴Agarose ∴Ethidium Bromide ∴TBE Buffer

⇒ A stock of 1% by concentration TBE buffer was generated. This will be used for all standard gels. Sterility with it is recommended, but not completely necessary.

⇒ Courtesy of Kevin, an instructional session on gel production was completed. A rough protocol was written, to which a refined version should be finished by the end of Friday 5.27.11. More practice with these methods would be of benefit to everyone intending on participating in lab work, however, so more sessions should be planned.

⇒ The transilluminator was tested. It works. Anyone running a gel should take a picture of it and upload it to the wiki.

⇒ The shaker/incubator/water bath was also tested. It's not clear if it genuinely requires water to exist in the bottom or if it can simply function as an incubator; when being set at 37°C, however, it seems to randomly rise above 38°C, then emit irritating beeping noises. Exactly how this device operates is still under investigation.

5/31/2011

Ben

Forever alone :'(

⇒ Gel protocol has been tested and a revised version will be uploaded.

⇒ 6 microliters of DNA ladder was used without dilution; this may be a bad idea. Please note the juice+ladder is in a clear centrifuge tube.

⇒ Protocol for gel viewer was tested. Please note the UV source itself is the true "transilluminator"; the entire machine should hence called the gel viewer.

6/1/2011

Ben

⇒ Gel protocol has been tested a second time without success. It appears mixing is absolutely crucial to all reagents, including the DNA; this was not stated in the protocol. No protocol currently exists for making 1% TBE buffer, which we are about to run out of; assistance with this would be appreciated.

⇒ Work has started on a do-it-yourself cold block for storing enzymes made out of solid ice. Current results look bad.

⇒ Preliminary designs for a transport vector that can carry surface display components is underway and is currently in the "argument" stage.

6/5/2011

Ben

⇒ Gel protocol has been tested a fourth time with success. Phusion from the freezerwas used in a rather haphazard way to generate PCR product, and a distinct band was observed. It should be noted that he TAQ solution is now dead for training purposes, but its buffer is receptive to other polymerases. This is completely useless information.

⇒ Enzymes from the Lin lab are now consolidated in the USB. We now also have a nice set of pipettes *borrowed* from the Lab.

⇒ Unbeknown to most, electric autopipetters exist which are portable and easy to use. They are in the cabinet. The current "plug in" compressor-based one is currently out of order as the filter is clogged; this is being investigated further.

6/8/2011

Chris, Narun, Ben

⇒ A digestion test was attempted on remains of the experiment from three days ago. This was completed as a training exercise. Results are still inconclusive. Leftover GFP was cut with XbaI and run on a gel next to a control of uncut GFP "just to see what would happen."

6/10/2011

Josh, Ben S, Chris, David, Ben P

⇒ Cryostocks for 10 different strains [2 Original INP Samples (non-standardized), GFP, linker(-80 and -20 samples), GFP-OmpA ligation (Resistance:K), Omp A, and a constituitive promoter] were taken out and grown overnight in the shaker for 16 hours. Stocks of old competent cells in the -80 were thawed, transformed with promoter J23199 from the 2009 iGEM distribution, plated, and grown overnight.

6/11/2011

Chris, Josh

⇒ All of the strains grown from the frozen stocks were cloudy. Control tube was clear. So, frozen stocks appear to be in good shape.

⇒ The plate for the transformed old competent cells did not appear to have any growth. Let incubate for another day.

⇒ A miniprep was performed on the ten strains grown from frozen stocks. 500 microliters of the original growth was stored in the fridge while 1.5 ml from each was used in the miniprep. The resultant DNA from the miniprep was stored in the freezer overnight.

6/12/2011

Alison, Ben P, Kevin

Note that Ben P is not alone for this post and no sad smiley faces should ensue.

⇒ Some basic lab prep was accomplished tonight

⇒ More 1xTBE solution was made (simple dilution of the 10x TBE with diionized water)

⇒ LB + AMP plates were made, along with LB + KAN plates

⇒ 2 400 mL LB solutions were made using 400 mL of diionized water and 18 g of Agar. These were autoclaved on the 20 minute liquid cycle. After they had cooled, 400 microL of AMP was added to one solution and 400 microL of KAN was added to the other solution. Once the bottles were cool enough to hold, the solutions were poured into Petri dishes to make plates. Sterile technique was used. Plates were allowed to rest and solidify overnight and were labelled and placed in the refrigerator to store. LB + AMP plates were labelled with blue marker and LB + KAN plates were labelled with red according to standards found online.

6/15/2011

Chris, Ben P, Alison

⇒ The QT comp cell production protocol was tested. 7 aliquiots of competent cells were produced (it was supposed to be five), which is rather unusual and will have to be investigated. Innoculation occured on the 14th at around 2200, leaving the growth time at around 17 hours. It should be noted the spectrophotometer is rather quirky and may require a larger amount of liquid than originally thought; this will be investigated as well, and the effected protocols modified.

⇒ 0.1M calcium chloride stock was produced a 1M stock-we don't have any solid CaCl2 laying around, so when we run out of the parent 1M stock we're done.

6/19/2011

Alison

⇒Sunday night lab prep consisted of mostly just cleaning. The liquid waste bottle was autoclaved, as were some other dirty glassware. Autoclave behaved very nicely. General tidying up of the lab was done and hopefully the rest of the time finds it satisfactory. It didn't appear that any stock solution or plates needed to be made. I will be out of town for next 2 Sundays, will maybe pass the baton to LJ for Sunday lab prep? Should probably actually talk to him.