Team:Lambert GA/labnotebook

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Revision as of 19:33, 21 June 2013

Lab Notebook

Date	Accomplishments
January 1-15	Met for the first time during class time as an Instructional Focus. Discussed project ideas and iGEM in general. Gave out protocols to study
January 16-31	Practiced transformations with p-GLO from Biorad. Learned about Bacterial Antibiotic resistance using Biorad Antibiotic Resistance Lab.
February 1-14	Discussed project ideas. Planned for visit to Georgia Institute of Technology, Styczynski Lab. Sold Pretzels for fund raiser.
February 15-28	Studied and took test over iGEM protocols and 3A Assembly. Finalized list of Biobrick parts of interest. Outlined initial ideas. Use K410000 to characterize cold shock heat generator.
March 1-14	Field trip to GA Tech on March 9 • Rehydrated J23119, J13002, J04450, K410000 • Miniprepped parts • Ran diagnostic gels • Sent parts for sequencing • Calculated concentration of DNA • Met with team members from GATech iGEM. Discussed the K410000 project. • New idea for project; isolate HybB and make it a Biobrick. GATech team will send their frozen stocks.
March 15-28	Worked on research for the new parts. Wrote Project proposal.
April 1-24	April 1st • Transformed again • Made Amp plates • Cataloged Gaucher 2010 box • Plated the HypB with RFP on Amp plates April 4th • Liquid culture results were analyzed • Took the HypB samples and mixed with Glycerol (Glyceroled Bio Bricks) and placed them in 80oC freezer • Streaked LB plates with HypB samples, promoter, and K410000 (bio brick) • Made more LB plates • Miniprepped hypB parts and K410 from Gaucher box • Calculated the DNA concentration • Calculated concentrations for sequencing • Wrote instructions for Cold Shock April 24th • Nanodropped with DNA concentrations • Ligation ( Protocol) • Digest of RFP, hypB, psBIC3, 410
May 1- May 14	• Transformation • RFP control and 410 RFP grew but only RFP has an expression • Ran transformation efficiency test with Protocol • Prep for experiment- plated 10 plates and inoculated 16 tubes • Experiment for temperature variation was successful but did not prove hypothesis
May 30th	Several of our members attended a safety training meeting, and are now certified in lab safety courses.
June 3rd	Designed primers and developed lab calendar
June 4th	Ordered primers
June 6th	We researched HybB and PSB1C3. Then chloramphenicol plates were made.
June 7th	We did a practice PCR reaction and ran a diagnostic gel to check practice our PCR. Primers came in, so we made stock solutions of a 1/10 dilution and a 1/20 dilution from the primers.
June 10th	We did a PCR of our HybB promoter and ran a gel. The gel showed that the temperature of our PCR was incorrect. Then we ran a gradient PCR with HybB promoter at deven temperatures ranging from 44.6°C to 53.3°C with the control at 54.5°C. Then we inoculated liquid stock with DH10 beta in order to purify genomic DNA.
June 11th	We ran a gradient PCR with HybB and R0011 as control. diagnostic gel showed the best temperature for PCR was 44.6°C and 53.3°C. On the gel we had bands around 400 base pairs, which is the expected length of our primers and part. We used QIAprep for PCR cleanup. We decided to combine 4 PCR reactions and purify to use in digestions and ligations. After purification HybB is 27.4ng/ul and control was 9.9 ng/ul.
June 12th	We met with Georgia Tech iGem team and heard their presentation and discussed cooperation and outreach. We did a double digest on HybB, PsB1C3, and control R0011. We used EcoRl remix and PST1. We blasted all of our parts to check all of the DNA sequences against the parts registry. We ligated HybB and PSB1C3, and a Control (R0011 and PSB1C3). In order to keep the backbones from reclosing we used Antarctic phosphatase to eliminate the phosphates on the ends. We also ligated 3 ratios of backbone to part. 1:1, 1:2 and 1:3. Ligase came to room temperature.
June 13th	We repeated the double digest because the ligase was suspect. We used Antarctic phosphatase to cut off the extra phosphates on the 5 prime ends of the backbone. We ligated the HybB promoter and the backbone and we included a control of just the backbone. Then we transformed the ligation mixtures from 6/12, 6/13, and a control. Our control was the backbone and R0011.
June 14th	We recorded the results of the plates. There were no colonies from the 6/12 ligations. Pictures of the 6/13 ligations have been included. 1:1 Ratio 1:2 Ratio 1:3 Ratio Antarctic Phosphatase (Just backbone) Control R0011 We did colony PCR using the HybB promers for 16 colonies, choosing the ligation mixture that was a 2:1 ratio of HybB to backbone. We set the thermocycler to 30 cycles annealing 50 degrees. Our elongation was 30 seconds. We did a diagnostic gel with 16 colonies and analyzed our results. Unfortunately, our colony PCR failed. We do not know the reason for the failure, but we suspect that a)we did not put the colonies in the thermocycler for long enough, or b)the cells did not contain our part.
June 17th	We repeated the colony PCR with 16 new colonies and a control. We set the elongation step to 45 seconds, thinking that the sequences may not have had time to finish. All other steps remained the same. Again, there were no expected or unexpected bands. Then, we inoculated liquid cultures with 5 different colonies from the plates of ligations.
June 18th	Then we miniprepped the liquid cultures. We found concentrations of DNA range from 79.5ng/ul to 114.5ng/ul. Then they were digested with EcoR 37C for 15 minutes. We ran diagnostic gel on the digests. We were expecting bands at 2070 bp for just PSB1C3 and 2463 for Colonies which contained HybB promoter sequence. We also ran them with a control of R0011.
June 19th	Amplification of HybB from DH10Beta and control. We used different concentrations of DH10 from 2 sources (ÜberDan's and ours), along with a control of R0011 in PSB1c3. The amplification failed, and the gel showed no bands. The pipetting of Taq was suspected to have caused the failure

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 <td>We ran a gradient PCR with HybB and R0011 as control. diagnostic gel showed the best temperature for PCR was 44.6°C and 53.3°C. On the gel we had bands around 400 base pairs, which is the expected length of our primers and part. We used QIAprep for PCR cleanup. We decided to combine 4 PCR reactions and purify to use in digestions and ligations. After purification HybB is 27.4ng/ul and control was 9.9 ng/ul.
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