Team:BYU Provo/Notebook/Metabolism/mayjune

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BYU 2014 Notebook

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Week of May 10th

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. Used Q5 polymerase and Q5 protocol.

  • Q5 PCR
  • 24.5 ul ddH20
  • 10 ul Q5 Buffer
  • 10 ul Q5 Enhancer
  • 1 ul Bla forward primer BI374
  • 1 ul Bla reverse primer BI375
  • 1ul dNTP’s
  • 1ul Template psB1A3 (resuspended with 10ul ddH20 from iGEM 2013 kit plate 5, well 1G)
  • 0.5ul Q5

May 9, 2014

Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using Gen-Elute Kit:

  1. Add 0.5mL column prep solution to column, spin at 12000 RPM 1 min. Discard eluate after 1 min.
  2. Transfer PCR reaction mix into the tube
  3. Add (5x’s volume) of binding solution to 1 volutm of PCR reaction and mix, 250ul
  4. Add to column, centrifuge at max speed for 1 min
  5. After 1 min discared eluate, but retain collection tube
  6. Add 0.5 ml of diluted Wash Solution to the column, centrifuge at max speed for 1 minute
  7. After 1 minute, discard the eluate, replace the column back in the collection tube. Centrifuge an additional 2 minutes to dry.
  8. Place the column in a clean tube. Add 50ul elution buffer, wait 1 min. Spin max speed 1 min. Remove column, voila!

Ran restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)

May 10, 2014

RD Protocol (repeated for each of the three samples)

  • 14ul ddH20
  • 5ul NEB Buffer 4
  • 0.5 ul 100x BSA
  • 30 ul DNA sample---(1 each of BBa_J23102, BBa_J23118, and PCR amplified Bla gene)
  • 1.5 ul each of SpeI HF, and XbaI

Incubated at 37C for 1.5hrs.

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 plasmid mini-preps 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.

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