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From 2014.igem.org

BYU 2014 Notebook Edit May June

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May 6, 2014

Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.

May 8, 2014

Set up PCR to amplify Bla gene. This is the iGEM link that eventually needs to be changed to *Q5 protocol link Used psB1A3 plasmid as template with primers BI374 and BI375.

May 9, 2014

Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using This is the iGEM link that eventually needs to be changed to *GenElute Protocol Kit linkRan restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)

May 10, 2014

Restriction digests were run on the following samples: Promoter part plasmids BBa_J23102, and BBa_J23118, and Bla gene PCR product. SpeI HF, and XbaI were used with NEB buffer 4 in this protocol for each of the three samples. This is the iGEM link that eventually needs to be changed to *Restriction digest protocol

Week of May 17th

May 13, 2014

Figured out that we didn’t want to use the promoter vectors that I digested. We tried using pIG91 from the freezer, but the RD gel did not show any DNA present. Also did This is the iGEM link that eventually needs to be changed to *Sigma-Aldrich plasmid prep protocol according to the Sigma-Aldrich kit, for new pIG91.

May 15, 2014

Assisted in setting up new RD of pIG91.

Assisted Nano-dropping the recently purified pIG91 plasmisds, all concentrations were approximately 100ng/ul.

Assisted in setting up ligation of digested pIG91 (treated with CIP), with the digested Bla. Both were digested using SpeI HF and XbaI. This is so that we can then have our part in the iGEM registry plasmid to allow for quick and easy future cloning.

Week of May 24th

===May 20, 2014=== Set up colony PCR. Chose 8 colonies from the transformation and streaked colonies onto another plate. Colonies 1-7 were white colonies. We chose colony 8 as a red colony to act as a negative control. Made a master mix (10x) the Taq PCR Protocol Taq PCR *19 ul ddH20 *2.5 ul Std. Reaction buffer *0.5 ul 10 mM dNTPs *0.5 ul each promer *0.5 ul Taq DNA polymerase *2 ul of boiled colony sample per tube. ===May 21, 2014=== Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights. ===May 22, 2014=== Did plasmid preps on overnights from colonies 4-7. Nanodropped: 206.1 ng/ul 260/280: 1.89 215.4 ng/ul 260/280: 1.89 400.2 ng/ul 260/280:1.85 170.0 ng/ul 260/280: 1.84 Set up restriction digests on New Plasmid (pIG91+BlaGenepIG102), and promoter plasmids to build final construct with promoter and the bla gene together. RD 5-221M (pIG102 cut with EcoR1 and Xba) *14ul ddH20 *5ul 10x NEB buffer cutsmart *.5 ul BSA *30 ul pIG 102 *1.5ul EcoR1 HF *1.5 ul Xba Incubate at 37 for 1.5 hrs RD 5-222M, and RD 5-223M Same protocol as above except: Enzymes used are EcoR1 HF and Spe1 HF Buffer 4 Used Two reactions, one containing promoter plasmid J23104 (5-222M), and the other J23111 (5-223M) Ran Low Melt Gel at 90V for 46 min. Each of the Plasmids showed up, which was good for pIG102, where we want that to be our vector. Other RD only saw the plasmids and not the promoter part which we are trying to insert into pIG102…. ==Week of May 31st== ===May 27. 2014=== Set up RD of promoter plasmids J23101, and J23106 Used 3 RD enzymes because we will not be able to run on low melt and get out promoter part, so we want to ruin the plasmid the promoter is in so that it doesn’t relegate. Used Enzymes EcoRI, PstI, SpeI RD 5-271M (J23101), RD 5-272M (J23106) *14ul ddH20 *5ul 10x NEB buffer 4 *.5 ul BSA *30 ul J23101, J23106 (in their respective tubes) *1.5ul EcoR1 HF *1.5 ul PstI HF *1.5 ul SpeI HF Incubate at 37 for 1.5 hrs To destroy enzymes reaction was then incubated at 80C for 20 minutes. Ligation of Vector and Insert Lig 5-273M (J23101 Promoter + gs5-221M) *6.5 ul ddH20 *1.5 ul lig. Buffer *1 ul DNA ligase *3 ul vector gs5-221M *3 ul insert RD 5-271M Lig 5-274M (J23106 Promoter + gs5-221M) *6.5 ul ddH20 *1.5 ul lig. Buffer *1 ul DNA ligase *3 ul vector gs5-221M *3 ul insert RD 5-272M Incubated at room temperature overnight Transformation *Melted ligations at 65 for a few minutes *2 ul of Ligation mix added to 25 ul dH5alpha *8 min on ice *Heat shock at 42 for 60s *Return to ice 3 min *Add 500 ul plain LB *Incubate at 37C for 30 min *Plate cells (250ul each) on LB+Cam, LB+Cam+Amp May 29, 2014 Took 8 colony samples from each of the LB+Amp+Cam plates and set up colony PCRs. Did according to Taq colony PCR protocol Used primer bIG307 pSB1C3 forward and bIG375 which is for Bla reverse. ==Week of June 7th== ===June 3, 2014=== Confirmed colonies via plating and colony PCR. Colony PCR showed colonies positive for colonies 1-4, 5, 6, and 8 for transformation of ligation 5-273M. However, on the plate only colony 8 showed up without RFP, thus I selected this colony as one that would be considered a final construct. Same process was repeated for transformed ligation 5-274M, where colonies 1, and 6 showed up on colony PCR, and where colony 1 was white—colony 1 was selected as a final construct. Overnights were set up and grown at 37C. ===June 4, 2014=== Plasmid Preps were ran on the overnight samples. ===June 5, 2014=== Plasmids were nano-dropped. *Lig 274M-1 415.2ng/ul and 260/280: 1.86 *Lig273M-8 515.6ng/ul and 260/280: 1.86 I also renamed these plasmids and submitted them to our BYU iGEM parts database, *Ligation273M-8 will be pIG105 *Ligation 273M-9 will be pIG106 ==Week of June 14th== ===June 10, 2014=== Looked at iGEM webpage, tried to figure out some code things…. ===June 12, 2014=== Set up Q5 PCR for genes NorB, NosZ *26.5 ul ddH20 *10ul q5Buffer *10ul q5Enhancer *1ul F Primer—(NorBF BI335, and NosZF, respectively) *1ul R Primer—(NorBR BI336, and NosZR, respectively) *1ul DNTP’s *0.5ul q5 *1ul Template (P. Aeroginosa) ===June 13, 2014=== RD of NorB and NosZ *5ul NEB Buffer 4 *0.5 ul BSA *~45ul DNA sample (NorB, and NosZ respectively) *1.5ul Xba *1.5ul SpeHf *Put at 37C for 2+Hours *Treated with CIP for 1hr Transformation According to protocol only used 4ul of ligation mix instead of 2.