Team:UChicago/Notebook - 2018.igem.org

6/14/18
-Got familiar with lab
-Made LB and normal YPAUD liquid media
-Labelled liquid media and left on bench shelf
-Make chloramphenicol plates
-250mg chloramphenicol; marked by CAM

6/15/18
-Made Arg- YPAUD liquid media
(-Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM (1.7g of CSM -Arg -Ura, 1.7g of uracil)
-Marked by 3 blue stripes
-Made Arg- YPAUD plates
    -Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM
    -Marked by three blue stripes
-PCR amplify arg-ars sequence
    -Total volume: 50µL
        -35µL water
        -10µL 5X phusion buffer
        -1µL dNTPs
        -2.5µL 10µM primer stock (forward and reverse arg-ars primers)
        -1µL pOW1 (17.6 ng/µL) DNA stock (0.8µL DNA, 4.2µL H2O)
        -0.5µL phusion
        -Stored in refrigerator next to PCR machine (labeled iGEM arg-ars PCR)

6/18/18
-Gel electrophoresis of arg-ars sequence (PCR amplified on 6/15/18)
    -Check if PCR was successful
    -Agarose gel:
        -40 mL 1X SB buffer, 0.32g agarose
        -Mixed and microwaved for 35 seconds
        -Added 2µL EtBr to agarose gel in flask
    -Agarose gel opaque and gray when fully polymerized
    -Sat in chamber, completely covered in buffer, until ready to run the gel
-Gel was unsuccessful, neither the ladder band nor the PCR product band could be observed
    -Suspected problem: samples not loaded into wells properly
        -Large smear of DNA across wells observed
-PCR amplified arg-ars sequence with 1 min per Kb (5 min since Arg-ars sequence is 4.86 Kb)
    -Labeled in blue marker

6/19/18 -PCR amplified arg-ars sequence with 30 seconds per Kb (2.5 minutes since Arg-ars sequence is 4.86 Kb)
    -Labeled in black marker
-Gel purification Arg-ars PCR products
    -Made 0.8% agarose gel
        -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr
        -Each well has 25µL of sample
    -Well 1 loaded with 1 min per Kb arg-ars PCR product
    -Well 3 loaded with DNA ladder---
    -Well 5 loaded with 30 sec per Kb arg-ars PCR product
    -Ran at 120V for 25 min (4.86 Kb sequence)
-Well 5 came out clear, however wells 1 and 3 were diffuse to be observed
    -Suspected problem: holding micropipette to the bottom of the well when pushing to the second stop, when pulling the micropipette out, dye doesn’t remain

6/20/18
-Gel purification Arg-ars PCR products
    -Made 0.8% agarose gel
        -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr
    -Well 1 loaded with 1 min per Kb arg-ars PCR product
    -Well 4 loaded with DNA ladder
    -Well 7 loaded with 30 sec per Kb arg-ars PCR product
    -Ran at 120V for 25 min (4.86 Kb sequence)
Bands were smeared
    -Michael: voltage likely too high, run at 90V; ladder lane did not have clear bands, try running the ladders against each other; the PCR product with 30 seconds per Kb produced very faint bands, use the protocol with 1 min per Kb
-Identified the (large and rather smeared) region where the arg-ars sequence was likely to be and excised it
    -Placed in eppendorf tube and added 450 µL of GQ buffwe
    -Placed in 50º bath for 10 minutes
    -Added 450µL of GQ buffer (solubilization buffer), centrifuged at 5.0k rpm for 1 min
    -Added 200 µL of PE buffer (wash buffer), centrifuge at 13.0k rpm for 30 seconds
    -Added 200µL of PE buffer (wash buffer), centrifuged at 13.0k rpm for 3 minutes
    -Inverted and set to dry for 3 minutes
    -Nanodrop detected no DNA (no peak at wavelength of 260 nm)
-PCR amplified arg-ars sequence (1 min per Kb)
    -Made two PCR products (each 50µL)
    -Used protocol outlined above
-Cleaned lab area

6/21/18
-Gel purification of arg-ars PCR product
    -0.8% agarose gel with 1X SB buffer, 2µL EtBr
    -Run at 105V for 30 minutes
    -Lane 1: arg-ars PCR product; lane 3: 100 bp ladder
    -Bands observed in gel were too smeared and also too short to be the desired sequence
-Simone’s suggestions
    -Not trying to PCR the entirety of POW1 (which is 4.86 Kb), only trying to amplify a 2.5 Kb section that contains ScARG4
    -Run PCR with annealing temp at 58ºC instead of 55ºC

6/22/18
-PCR amplified arg-ars sequence with an annealing temp of 58ºC
-Gel electrophoresis of arg-ars PCR product
    -0.8% agarose gel with 1X SB buffer and 2µL EtBr
    -105V for 35 minutes
    -Lane 1: 1 Kb ladder; lane 3: arg-ars PCR product; lane 5: arg-ars PCR product
    -Lane 5 did not come out clear
    -Ladder too bright, added too great a volume to the well (20 µL instead of 5µL)
-Gel purify arg-ars PCR product
-No DNA detected by the nanodrop

6/22/18
-Did diagnostic work on the PCR reaction performed by RF
    -Suspected cause 1: Template was at 18.2 ng/µL instead of the 96.5 labeled on the tube
        -Sample also has a 260/230 of 1.27 indicating EtOH contamination inflating this value
        -Likely that there is not enough template to successfully create PCR product
        -Only 2µL remain anyway, so need new pOW1 to proceed anyway
    -Suspected Cause 2: Primers are not specific enough. Forward primer’s last 5 3’ bases match to 7 locations. One of these forms a 600 bp product that is consistent with the lower band. Can extend the primer 5 bases to create a specific primer that only binds to its target site.
-To grow more pOW1, prepared an overnight culture of PPY12 as Allison instructed the plasmid to be grown in yeast

6/23/18
-Talked with Dr. Glick
    -Indicated that cannot grow more plasmid in the PPY12 overnights as they need to be grown in bacteria
    -Transformed 1µL of remaining pOW1 plasmid into DH5α cells. Plated onto an LB+Amp plate (See below)
-Created 20 LB+Amp plates following Glick lab plate recipe. Plated both my sample on 1 and a negative control on another. Did not have a strain that I knew had Amp resistance already, so I was unable to do a complete positive control.

6/24/18
-LB+ Amp Plate result
    -Negative control had no growth indicating the antibiotics are in sufficient quantity to kill non antibiotic resistant bacteria
    -pOW1 sample had too many colonies to count indicating a successful transformation was likely (Had no positive control, so cannot be fully certain until attempt to isolate the plasmid is performed)
        -Inoculated 5 overnight liquid cultures (4 for plasmid harvesting and one to make a glycerol stock and future positive Amp control)

6/25/18
-Miniprep Liquid cultures A→ D
    -Concentrations
        -A: 748.9 ng/µL
        -B: 573.3 ng/µL
        -C: 527.9 ng/µL
        D: 554.2 ng/µL
-Made glycerol stock of Liquid culture E
6/26/18

-PCR amplified arg-ars sequence from pOW1 samples B-D
    -Want ~20 ng of the plasmid DNA (these dilutions result in pOW1 concentrations of 20 ng/µL)
A: 0.8µL pOW1 A, 29.2 µL H2O
        -B: 0.8µL pOW1 A, 23.0 µL H2O
        -C: 0.8µL pOW1 A, 21.1 µL H2O
        -D: 0.8µL pOW1 A, 22.2 µL H2O
-Made glycerol stock of liquid pOW1 culture E
-Gel electrophoresis of arg-ars pOW1 A-D PCR products
    -Lane 1: 1 Kb ladder; lane 2: pOW1 B; lane 4: pOW1 C; lane 6: pOW1 D
    -Ran at 110V for 30 minutes
        -Gel imaging showed only a smaller band around 100 bp for lane 2 (pOW1 sample B)
            -Suggests primer issue; will address in team meeting tomorrow
-PCR amplified arg-ars sequences from pOW1 sample A

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.