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

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