Team:Baltimore US/JulyGroupNotebook

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Group Notebook for July 2010

@ Roberts Lab / Towson In Attendance: Dr Roland Roberts, Dr Liz, Dr Schiefele, Patrick, Ryan, Duke

Dr Liz introduced this group of iGEM folks to Dr Roland Roberts. Dr Roberts is willing to have a small cadre of the iGEM Team Baltimore work in his lab. Because Dr Roberts is not officially an iGEM Advisor, we are going to be especially considerate about using his lab space. We are not going to have any more people come into his lab space at this time. There may be room for one more person to work in the Roberts' Lab at a later date.

Dr Roberts has a very disctinct protocol in his lab, and he was kind enough to distribute Lab Instructions to all of us -- these are what he gives his own students when they first comoe into his lab. We are very grateful for Dr Roberts' support, and we will show that by being especially sensitive to his lab policies and procedures. Dr Roberts keeps a sterile working environment. PCR reactions and cloning are always done under the hood. His lab only uses filtered tips. Dishes do not stack up in his sink. We will help do the autoclaving. Ethidium Bromide is done in only one area of the lab - it is stained into gels after they are run, rather than put into the gel agarose. The reasons for all of Dr Roberts' protocols are completely obvious. (Even if they weren't, we would follow them anyway!)

We will have to provide ALL of our expendables - from gloves, tape, felt-tips, foil for autoclaving, tissue wipes, clips and storage bottles, to all reagents we need for reactions. That means gel agarose, molecular ladder, restriction enzymes, ligase, competent cells, antibiotics, cell culture plates and agar, DNA oligos, primers, dntp's, polymerase, BSA, buffers for PCR and Restriction digestion reactions. We also need to bring over a gel apparatus or two (he has ample power supplies), pipetmen, filter tips, tubes of all sizes, work blocks for holding tubes, chemical jars (orange lids) if we need to mix up own own solutions. Other needs will become apparent as we begin to perform experiments in the Roberts lab.

We do not have to provide our own TAE, TBA or paper towels. These items are cheap enough that Dr Roberts can let us use his. We will not bring ethidium bromide for now. Maybe later.

We may use Dr Roberts' glassware. If you break it, Dr Liz buys it. Please be careful! We have access to PCR machines, rockers, water bath, centrifuges, spinners, cold centrifuge, fridge, freezers (both -20 and -80), microwave, autoclave, and the gel imaging system. The cell culture room is on the 4th floor of Smith and is open all the time for anyone's use.

We may also use the PC's in the lab for lab-related work or checking email. (Please do not update your facebook account on Dr Roberts' PC's!). He has two pipet stands - one has been spoken for by Dr Liz. Sr Jim Saunders, Director of the MB3 Program, has gotten lengthy wish lists over the last few days from Dr Liz, and he has already provided storage boxes for tubes. He emailed Dr liz yesterday and said he has more stuff. Dr Saunders is sure to be a great resource.

In Dr Robert's lab, it goes without saying that Dr Roberts' say is final. Otherwise, Dr Liz's say is final. It is her reputation on the line at the Towson Lab space, and anything the iGEM team does will reflect directly on Dr Liz. Anyone who cannot work under that caveat should bow out now from the Towson Lab space.

On a more upbeat note, the Towson Lab will have results to report soon, we hope!

July 27th, 2010

@ Burkett Lab Day Shift: Patrick, Robert, Duke and Ryan

Running restriction digests on ANN parts for validation. Problem with <partinfo>J23030</partinfo>: restriction digest shows only a faint band of genomic DNA in lanes 2 & 3 (cut, uncut with Spe1):

http://imgur.com/ePNEO.jpg

Will run a larger gel tomorrow with all ANN parts to confirm.

--Pon 22:40, 27 July 2010 (UTC)

Ryan ran the transformations for the PoliColi Ignition Ligation. (<partinfo>R0010</partinfo>, <partinfo>B0034</partinfo>, <partinfo>PsB1C3</partinfo>)
https://static.igem.org/mediawiki/2010/c/c2/BbPart_%28Ignition-R0010%2CB0034%2CPsB1C3%29Transformation.pdf
When we return to the lab on thursday, cell plates must be checked from incubator. Looking especially for results on RXN plate, sample should be run for electrophoresis sizing after additional culturing.

July 26th, 2010

- Burkett Lab Attendence: Robert, Duke, Ryan, Patrick

Ryan ran the Photospectrometer analysis on the Bb DNA that's been cultured for the ANN and PolIColi ignition components. Results are as follows...

<bbpart>J01001</bbpart> A260 = 1.03 Concentration= 51.57 ug/ml Concentration = 0.05 ug/ul
<bbpart>J01003</bbpart> A260 = 2.37 Concentration = 118.55 ug/ml Concentration = 0.12 ug/ul
<bbpart>J23007</bbpart> A260 = 1.33 Concentration = 66.72 ug/ml Concentration = 0.07 ug/ul
<bbpart>J23008</bbpart> A260 = 2.52 Concentration = 125.74 ug/ml Concentration = 0.13 ug/ul
<bbpart>J23022</bbpart> A260 = 1.87 Concentration = 93.46 ug/ml Concentration= 0.09 ug/ul
<bbpart>J23030</bbpart> A260 = 3.11 Concentration = 155.94 ug/ml Concentration = 0.16 ug/ul
<bbpart>J23031</bbpart> A260 = 0.14 Concentration = 7.11 ug/ml Concentration = 0.01 ug/ul
<bbpart>J23032</bbpart> A260 = 2.01 Concentration = 100.36 ug/ml Concentration = 0.10 ug/ul
<bbpart>J23036</bbpart> A260 = 2.08 Concentration = 104.07 ug/ml Concentration = 0.10 ug/ul

<bbpart>R0010</bbpart> A260 = 1.44 Concentration = 72.19 ug/ml Concentration = 0.07 ug/ul
<bbpart>B0034</bbpart> A260 = 2.55 Concentration = 127.36 ug/ml Concentration = 0.13 ug/ul
<bbpart>B0015</bbpart> A260 = 2.43 Concentration = 121.23 ug/ml Concentration = 0.12 ug/ul

July 23rd, 2010

at Towson Campus In Attendance: Liz, Roland Roberts, Jim Saunders

Dr Liz met with Dr Roberts and was given the tour of the lab space and equipment available for Team Baltimore to use if a subgroup wants to work at TU. All the usual equipment is available except Dr Roberts want Team Baltimore at TU to bring their own set of pipetment, and of course, supply its own needs for expendable items. Dr Jim Saunders has taken a copy of Team Baltimore at TU's wish list, and says he can provide a set of pipetment, and tips/tubes without much trouble. We also need to take a gel electrophoresis apparatus, because Dr Roberts has a limited supply and other students use them frequently. He does have an extremely large one he said we could use.
Dr Roberts wants to meet with the students who wish to have access to his lab, sooner rather than later. It is important that Team Baltimore bring reagents over to the Roberts Lab and begin to use some of the space Dr Roberts has set aside for us, in order to demonstrate that we are serious about using his lab. Anyone who would like to work at the TU site sometimes should contact Dr Liz immediately.
There is a DNA sequencer and a high-throughput DNA seperator that is accurate to 1 bp for oligos between 100-650 bp long. If Team Baltimore wants to use this machine, that is possible. In that case Dr liz will talk to her Dean at TU to acquire funds to help share the cost of using it.

July 20th, 2010

In Attendance: Gary, Bernadette, Tom, Patrick, Ryan
During day shift, Robert finished working on PCR optimizations. While Patrick and Ryan, troubleshot their primers and put the order in so that they can begin PCR reactions when they return on monday.
Bernadette concentrated on her Ligation reactions for the PolIColi project.
Ligation reaction (COLOR CODED RED)included: 6 ul R0010 promoter, 6 ul B0034 RBS,
2 ul PsB1C3 chloremphenacol resistant plasmid,2 ul buffer, 3 ul H20 & 1 ul ligase enzyme
For comparison, I also completed a 'No Insert' group (COLOR CODED TEAL/BLUE)using 2 ul plasmid, 2 ul buffer,
15 ul H20, 1 ul ligase
In addition a 'No Enzyme' group (COLOR CODED DARK GREEN)was completed that included 6 ul R0010 promoter,
6 ul B0034 RBS, 2 ul plasmid, 2 ul buffer, 4 ul H20
The 3 groups were incubated at 16 degrees C overnight

Gary got caught up on project status and development.

July 19th, 2010

In attendance: Miles, Patrick, Robert, Duke, Liz, Tom, Bernadette, Steven
Steve transformed <partinfo>J23030</partinfo> as it was the only DNA from last week's transformations that did not develop any colonies.
Bernadette began restriction digestions for the Bbparts for the PoliColi project.
Part #'s
<partinfo>R0010</partinfo> The LacI Promoter
<partinfo>B0034</partinfo> The Strong RBS
<partinfo>PsB1C3</partinfo> A Chloremphenacol Resistant Plasmid Backbone
<partinfo>B0015</partinfo> The Double Terminator
<partinfo>R0010</partinfo> and <partinfo>B0034</partinfo> are meant to be ligate into the <partinfo>PsB1C3</partinfo> as all the initial PoliColi Bbparts had a native Amp resistance and one had Amp and Kanamyacin.
The <partinfo>B0014</partinfo> will be ligated with the PoliColi NewPart once the Overlap Extension Point Mutation PCR is completed.

Restriction digestion included the following: R0010 Promoter (COLOR CODED BLUE): 5 ul DNA, 5 ul buffer#4,
37.5 ul H20,cuts made with 1 ul EcoRI and 1 ul SpeI enzymes, and .5 ul BSA The result will be the insert
In a separate reaction B0034 RBS (COLOR CODED PURPLE): 5 ul DNA, 5 ul buffer #4, 37.5 ul H20, 1 ul XbaI,
1 ul PstI,.5 ul BSA
The 3rd reaction PsB1C3 plasmid(COLOR CODED LIGHT GREEN): 5 ul DNA, 38 ul H20, 5 ul buffer #4, 1 ul EcoRI,
1 ul PstI, no BSA
Incubated overnight 37 degrees C

Ryan and Patrick began working on Primer design for their various PCR projects.
Miles showed off initial development of Gel Electrophoresis Kit and Power Supply. Discussed PCR construction with Tom, as well as introduction to Oligonucleotide synthesis.

July 12th - 15th, 2010

Dayshift Lab Techs began DNA Preparation of various identified BbParts. Robert and Duke finished with Boiling Prep.
Evening Lab intermittently closed with Tom unavailable for lab supervision.

July 8th, 2010

In Attendance: Gary, Patrick, Robert, Lisa, Liz, Steven Strategy Meeting in spare room. Patrick outlined strategy of ANN (Artificial Neural Network) based on Berkeley's previous work. Discussed use of conjugation plasmids for weight assessment.

July 6th, 2010

Can't quite remember back far enough to state what occurred. Got to remind everyone to update the notebook wiki page as during the evening's in question.

July 1st, 2010

Ryan didn't make it, but I hear something happened.