Dahlquist:SURP 2019 Schedule

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Week 1

Week 2

Alice

  • Alice updated protocol with links to archived files on GitHub.
  • TO DO (Alice): Organize the main stem results for each strain so that the same profile is all down one column, add it to the summary PowerPoint.
  • TO DO (Alice): For profiles that are the same across strains, she will determine whether the Gene Ontology terms are the same or different for the profile. She can use Microsoft Excel to do this based on a protocol on this page. Searches for a tool that can compare two GO term lists turned up the following:

Alyssa & Susanne

  • Alyssa created protocol page for Preparing Cheek Cell Lysate based on the BIOL 478 Lab 5 prep list and protocol.
  • Alyssa and Susanne prepared 24 tubes of 0.9% saline.
  • They prepped cheek cell lysate from 5 individuals, three of whom were the same from the Spring BIOL 478 class. These were performed as repeats so that we could verify that we got the same results as before.
  • They performed the PCR reaction on those samples + lysate from the three matching individuals from BIOL 478, ran the gel, and purified the PCR products according to this protocol.
  • The three control PCR products were sent to be sequenced.

Week 3

Alyssa & Susanne

Completed

  • Set up search alerts for PubMed for several variations on key terms
  • Analyze sequencing results: Susanne updated her notebook and uploaded edited text files to Box. Blast2Seq to reference sequence on the protocol page.
  • Set up cited reference alerts for key articles through Web of Science.
  • RFLP analysis complete for the PCR samples prepped during Week 3.
  • TOPO cloning of 448 bp fragment TT sample, CC sample.
    • Prepare LB liquid media and plates for cloning (6/4/19).
    • Perform control PCR from TOPO kit (6/5/19).
    • Perform TOPO cloning reaction on vector-only control, vector+control insert, TT sample, CC sample (6/5/19).
    • Transform with the above + pUC19 control plasmid + negative control (water) (6/5/19).
    • Pick transformants: streak on fresh plate (6/6/19).

Week 4

Alice

  • Began modeling experiments
    • Variable inclusion of strain data for db5
      • Made a list of experiments in Excel to keep track.
      1. wt-only
      2. wt + each strain individually
      3. wt + 2 strains
      4. wt + 3 strains
      5. wt + 4 of the five deletion strains (in other words, leaving one deletion strain out)
    • completed runs, compiled data
    • Made LSE:minLSE bar charts, heat maps

Alyssa & Susanne

  • Inoculate O/N cultures (Sunday 6/9/19).
  • Mini-preps (gel), 6/10/19
  • PCR/gel to confirm insert (all had inserts)
  • Submitted for sequencing 6/11/19 (15 to 20 μL of purified plasmid and PCR DNA at concentrations between 50-100 ng/μL for Laragen)
  • Analyzed sequencing results and got a verified clone (CC3 and TT1) for each genotype

Week 5

Alice

  • Analysis of variable inclusion of strain data runs
    • Bar charts for P's and b's
    • Looked at expression plots and GRNsight networks. Maybe the LSE:minLSE ratios are getting bigger for the dGLN3 and dZAP1 data because the expression is more divergent from the other strains and the model has to balance matching all datasets. In this view, an increasing LSE:minLSE ratio is not necessarily bad.

Alyssa & Susanne

TO DO

  • Computer work:
    • Add representative reference for each SNP so we can connect it back

Completed

  • Computer work:
    • Scan through the Web of Science results for the Tishkoff paper. Status?
    • Make sure all the cited references for the Labrie et al. (2016) paper are in the bibliography
    • Look at dbSNP to make a list of SNPs in the region, then search for their related papers in PubMed.
    • Make a list of populations/countries/regions where LP genotyping has been carried out. Look in OpenSNP, too.
    • Convert list of SNPs on wiki to Excel (file already on Box, edit with Excel online)
    • Try to find rs ID's for the SNPs that don't have them yet.
  • Lab work:
    • Additional mini-preps of CC3 and TT1 to build plasmid stock.
    • Resequence CC3-M13 reverse to get better trace. Also did M13 forward and Lac-13910-for-EM and Lac-13910-rev-EM for comparison
      • Only one of the four sequencing reactions worked
    • Perform restriction digests on CC3 and TT1 plasmids to cut once in vector and once in insert
      • StuI (USB Buffer M) cuts once in insert
      • BssHI cuts once in vector
      • DraI (USB Buffer M) cuts both vector and insert (thought only cut insert at first
        • Cuts after 2340, 3032, 3051, the 19 bp fragment between 3051 and 3032 will not be detectable, so would look like 2 fragments produced.
        • DraI generates 169 + 279 bp fragments out of 448 bp insert
      • StuI linearized plasmid as expected, but generated a size ~5200 bp instead of the expected 4379
      • BssHI only did a partial digest
      • DraI generated extra fragments
      • So redid with SpeI which cuts once in vector and once in insert, vector fragment still looked too big
      • KD: A call to technical support to ask about the vector size issue was unhelpful. They just said to do the control PCR reaction and clone it.
        • KD: Re-transformed with leftover TOPO cloning reaction that had been stored at -20°C.
        • KD: Did all four reactions, vector-only, control insert, CC, and TT inserts
        • KD: Got transformants for all four reactions and re-streaked vector-only (blue) and control insert (white) colonies
        • KD: Forgot that control insert needed LB + kanamycin instead of ampicillin
    • Do PCR with Lac-13910-for/rev-EM primers on
      • Cheek cell lysate for CC, CT, TT genotypes (got expected 448 bp bands)
      • CC3 and TT1 plasmids at 1:10 and 1:100 dilutions of ~70 ng/μL stock (got expected 448 bp bands. 1:10 and 1:100 dilutions were indistinguishable, so can use the 1:100 stock (or maybe lower). PCR products were purified in anticipation of FaqI digest in Week 6 when new enzyme stock available.
      • Attempted to use other set of primers on cheek cell lysate to see if SNPs always match at those two positions. PCR did not work very well, generating multiple fuzzy bands. Probably need to optimize either the annealing temperature or the MgCl2 concentration.

Week 6

Alice

  • Continued to analyze variable inclusion of strain data model runs
    • Further analysis of expression plots in terms of MSE (also maybe compute MSE:minMSE ratios)
  • Lab checkout
    • Made sure that electronic notebook is complete
    • All files uploaded to GitHub, Box, and/or OWW.

Alyssa & Susanne

  • Human Subjects Online Training: follow instructions to register here.
  • Digested 13910 PCR from Week 5 with FaqI and got expected bands with both cheek cell lysate samples and plasmid template samples.
    • New FaqI enzyme works
    • Plasmids can be used successfully for controls in RFLP, regardless of what weirdness is going on with the size of the vector
    • Ready to rock in the Fall!
  • Did plasmid miniprep for pCR2.1 TOPO vector to have vector-only control for restriction digest
    • Performed EcoRI restriction digest on vector-only plasmid control and on CC3 and TT1 plasmids.
    • EcoRI cuts on both sides of the insert site, so should linearize the vector and cut out a ~448 bp fragment on the vectors that have insert.

Future Directions

Alice

Alyssa & Susanne

  • qPCR genotyping reaction for 13910 C/T

Journal Clubs

Week 2 (May 28, 2019)

Week 3 (June 3, 2019)

Week 4 (June 10, 2019)

Week 5 (June 17, 2019)

Week 6 (June 25, 2019)

SURP Workshops

  • Thursday, May 30, 12:00-1:00, UH 3999 (McIntosh Center): Making Research Relevant Across Cultures (Alyssa, Susanne, Dr. D)
  • Wednesday, June 5, 11:30-12:30, WHH 118 (Library): Are You LinkedIn, or Left Out? (Alyssa, Susanne)
  • Thursday, June 20, 12:00-1:30, WHH 117 (Library): Abstract Writing Workshop (Susanne)
  • Wednesday, June 26, 12:00-1:00, WHH 117 (Libary): I am Good at Stuff: Crafting an Effective Resume (Alyssa)
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