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Team:BYU Provo/Notebook/Metabolism/febapr
From 2014.igem.org
BYU 2014 Notebook |
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Week of May 3rd
April 29, 2014
May 1, 2014
May 2, 2014
Week of May 10th
May 5, 2014
This week in the lab we started our experiment to remove the sera gene from N multiformis. We attempted to gain access to the genomic DNA of N multiformis by boiling the organism for 5 min to lyse the cell, this technique has worked on similar organisms in the past and due to its simplicity we opted for this approach. After boiling we added our primers and proceeded to perform PCR. Then run our PCR product on a gel. We did this by adding our DNA samples (both forward primers and reverse primers) and a DNA ladder of known size to an agarose gel that had been stained with ethidium bromide and electrophoresing for 30-40 min. After isolating our band of DNA we purified it using a freeze and squeeze method. This method involves excising a band of DNA from the agarose gel and the gel slice cut into small pieces and placed into a micro centrifuge tube. This tube is then placed in a -20C freezer for 20 minutes then removed and immediately centrifuged at 12,000 for 5 minutes at room temp. Agarose debris is will be forced to the bottom of the cup and our now purified DNA is floating on top ready for removal.
May 6, 2014
We finished out the freeze and squeeze experiment. We took the agarose gel from the freezer that held our PCR product and centrifuged it for 5 min at max speed the added to the following protocol for a freeze and squeeze to get our purified product. Freeze and Sqeeze 2ul fragment one from PCR 2ul fragment two from PCR 4ul 5x buffer 4ul 5x enhancer .5ul dNTP .5ul primer on left side of left homology block .5ul primer on right side of right homology block .2ul Q5 enzyme QA 20uL ddH2O Because Desi said that doing a freeze and squeeze was probably an unnecessary step we also ran a normal where we do not do a freeze and squeeze, rather we run straight to the SOEing part of it. SOEing protocol 1ul fragment one from PCR 1ul fragment two from PCR 4ul 5x buffer 4ul 5x enhancer .5ul dNTP .5ul primer on left side of left homology block .5ul primer on right side of right homology block .2ul Q5 enzyme QA 20uL ddH2O We got our SOEing product and ran it on a gel (picture taken and to be included once we scan it). We also performed a restriction digest and plasmid prep.
May 8, 2014
May 9, 2014
May 10, 2014
Week of May 17th
May 13, 2014
May 14, 2014
May 15, 2014
Week of May 24th
May 20, 2014
The past week I have spent approximately 6+ hours researching funding opportunities for our team and estimating costs. Opportunities include • Rathnau Instituut Grant • Departmental Funding • Crowd founding initiatives o I have started to build a profile on Experiement.com • Various Donors Verified our clones via colony PCR We did this by taking samples from 8 colonies to test for our clone. Alongside the 8 colonies we also ran a negative control (just the plasmid with no insert) and a positive control (our original soeing product) Protocol: 16.5 DDH2O 2.5 REdtaq Buffer 1 DNTP 1 Primer A 1 Primer B 1.25 Redtaq 2 Boiled template (colony) These we PCR’d and wil check on Thursday. 13May2014 – 19May2014 The planned schedule Week 1 Ligation, Transform Week 2 Conjugate (long time) • Grow the transformed ecoli (s17) • Grow N multiformis • Mix together, turn off the lights, but on some Barry White and wait Week Work on getting funding * experiment.com • Fancy black card man• Week4 Select for knock out with Kanamycin Select with sucrose Grow in serine rich, low, and no environments Thursday: Today we took tehe product from out ligtion and transformed it. (protocol to be added) We made 3 tubes Tube 1: 200uL s17, 1uL of 93.1 ng/uL mini-prep pSR47s Tube 2: 200uL of our ligation. Tube 3: 300ul of LB, 90ul ddH2O, and 1ul of S17 • S17 is the bacteria that makes our suicide plasma grow. All 3 tubes were placed on a shaker at 37 degrees C. Tuesday: Today (Tuesday) we did ran a gel of the plasmid prep (low melt) of the plasmid digest and the PCR digest (pics to be uploaded) We also did a mini-prep with the pSR47s plasmid. And a ligation
May 21, 2014
May 22, 2014
May 23, 2014
Week of May 31st
May 27, 2014
May 28, 2014
May 29, 2014
May 30, 2014
Week of June 7th
June 2, 2014
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June 3, 2014
June 4, 2014
June 5, 2014
June 5, 2014
Week of June 14th
June 10, 2014
June 12, 2014
June 13, 2014
Week of June 21st
June 17, 2014
June 19, 2014
Week of June 28th
June 24, 2014
We grew cultures of our final product (this time using the proper primers) and perfored a clone verification. We discovered that we had bands at 1000 base pairs! Yay!! That means that it works and we are not failures. Now we need to sequence the DNA and we plan on doing plasmid prep as soon as someone returns the missing reagent to the plasmid prep box. We presented to the class on Wednesday and also ran a plasmid prep now that we know that reagent 1 is kept in the fridge ☺ Desi was totally awesome and did our sequencing for us so thanks Desi. The results are shown below RESULTS SerA-2F AWWGRWCGMWTCTCTWTSYKTCSACGGTATCSATAAGCTTGATATCGAATTCCTGCASCCCGGGGGATCCGGCGTAATGGMACWCATGATCAAAACGGCGGCGGATGCCGAAGCGGCCGTAAACKCAGTATATTACCCTCCACGCGGACAACGTGGAGTCGGGCTTGCACGCGCCCAGGGGTATGGTGCGCGATTTCAGCAATATCGCCACTGGCTGGAAGAAAATGCCGTCATCATTGCCATGATCGAGCATATCGATGCAGTCGATGCCATCGATTCGATTCTTTCCGTTCCGGGAATCGATGCTTATATCATAGGTCCCTACGATCTTTCAGGATCGCTCGGCCGTCCCGGAGAACTCGCTGATCCGGAAGTCCAGGCTGCGGTAGAACGGGTAAAAGATGCCGGGCGACGCGCGGGCAAGGCAGGCGGCATTCATGTAGTCGAACCCAATCCGGAACAATTGCGCCGCAATATCGAGGCGGGCTTCAGTTTTCTTGGCTATGGCCTGGATATCCGCATTCTCGATACCGTCTGCCGCAGTCATCTGCAAAACATCAGGGAAGCTCTATGAACAAACTTGCGATTTCCACCTCGTCATTCGATGTCAGCATCGAAGCCGCTATGGCTCGCTCTATGACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTRTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAWTTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAMCCTGTCGTGCCAGCTGSATTAATGAATCGGCCAACGCTARAWTTCCCATGTCAGCCGTTAAGTGTTCCTGTGTCACTCAAAATTGCTTGARAGGSTCTAARGGCTTCTCARTGCGTTACATCCCTGGSTTGTKGTCCRCACCGTTAAACCTTAAAAGCTTRARAGGCTTATATATCTTTTTTCTWAWAAAMCTAAAACCTARRRGCTATTAGTTGCTGAATTTATATAAGTTAATGGTCARACATGAGAGCTAAGWASGGGAAAMTGGARAGCGTAGTACGTAGCCATGAAAGCTTAGTACGTAGCCAGGAGGGGTTAARGTCGTACATGAAGCTTAGTACGTAACCGTGAGAGCCTATAAMCGTGAAAGCGTGGATARGCCGGAT
June 25, 2014
--CB TR-- Today we prepared our presentations. We then gave them.
June 26, 2014
--CB TR-- Today we performed a plasmid prep (see protocol in common procedures section)
