Difference between revisions of "Team:RHIT/Notebook"

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<h2>6/27/16</h2>
 
<h2>6/27/16</h2>
  
 +
<ul>(XX) Goal: Redo digest and harvest TU and mls</ul>
 +
<ul>Major steps:</ul>
 +
    <ul>
 +
          <ul>Cut mRPS12 TU with EcoRI/PstI</ul>
 +
          <ul>Cut Mrps12 mls with EarI/SpeI</ul>
 +
    </ul>
 +
<ul>Gel order: L,TU1a, TU1b, TU2a, TU2b, mls1a, mls1b, mls2a, mls2b</ul>
 +
<ul>Gel results:</ul>
 +
    <ul>
 +
          <ul>harvested TU bands from TU2a and TU2b (2 bands per tube)</ul>
 +
        <ul>
 +
                          <ul>TU gel extract = 0.363 g</ul>
 +
        </ul>
 +
          <ul>harvested four mls bands (2 bands per tube)</ul>
 +
        <ul>
 +
                          <ul>mls gel extract A = 0.342 g</ul>
 +
                          <ul>mls gel extract B = 0.255 g</ul>
 +
      </ul>
 +
    </ul>
 +
</ul>
 +
<ul>Other activities:</ul>
 +
      <ul>
 +
          <ul>QIA gel extraction kit</ul>
 +
          <ul>Used nanodrop to test DNA amount:</ul>
 +
            <ul>
 +
                          <ul>TU = 3.1 ng/
 +
                          <ul>mls A = 41.2 ng/
 +
                          <ul>mls B = 27.7 ng/
 +
            </ul>
 +
          <ul>*according to nanodrop readings, TU needs to be precipitate while we run NEBuilder on mls A and mls B</ul>
 +
</ul>
 +
(HC) Ran NEBuilder on P413 GPD and 16-1 fragment.
 +
1μL DNA
 +
3μL fragment
 +
10μL NEBuilder HiFi DNA Assembily Master Mix
 +
6μL Sterile DI water
 +
 +
 +
(JM/CH)
 +
 +
The first thing we are doing is running the blunt end ligation protocol using NEB Quick Blunting Kit and the NEB Quick Ligation Kit
 +
 +
This was done with 3 tubes of 416 CYC1 and 3 tubes of 426 GPD
 +
 +
Next we did a transformation on one of the 416 CYC1 and one of the 426 GPD
 +
 +
Those are spread on plates and are now incubating so we can use them tomorrow
  
  

Revision as of 18:28, 18 August 2016

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NOTEBOOK.

I should probably put some Table of Contents up here, so one could easily skip from day to day... at any rate, here's the current info dump.

Heads-up: I know there aren't circles on the list. This is apparently harder to do than I had envisioned?

6/13/16

    (HC) Made LB-Amp plates
      450ml of DI water
      5.0g tryptone
      2.5g yeast
      5.0g NaCl
      7.5g Agar
    This was then adjusted to a pH of 7.0 using NaOH. It was then autoclaved. 2.5mL of 10mg/mL of Amp for a final concentration of 50 μg/mL was achieved.


6/14/16

    (HC) made CSM -His plates


6/15/16

    (XX) Goal: Run a gel to check whether pSB416 GPD has PstI site removed (diagnostic gel)
      1. pSB416 GPD cut wit AvaI
      2. pSB416 GPD cut with PstI
      3. p146GPD cut with AvaI
      4. p416GPD cut with PstI
    Major steps:
    Digest:

    6 sterile water, 1 CutSmart buffer, 2 DNA, 1 enzyme (AvaI or PstI-HF)

    Gel Order: L, 1, 3, 2, 4
T--RHIT--61516xx.jpg
    Expected results on this diagnostic gel:
      1. No cut because no AvaI site in pSB416 GPD
      2. Should only see one cut because only one PstI site
      3. Should see one cut
      4. Should see two bands
    Experimental results:

    Gel results are not good; need to rerun this diagnostic gel

    Other Activities:
      Created mRPS12+/KanMZ- (parent) and mRPS12-/KanMX+ (mutant) master plate on YPD
      Replica plating YPG (Should this be YPD???) , YPG, -His, -Ura, -Ura+Glycerol, YPD+Paro, YPG+Paro


6/16/16

(HC) Ran a boiling and spin prep on P413 GPD

    Boiling Prep Procedure:
      1) Pellet cells from 1.5mL of an overnight L-Amp culture of transformed E. coli cells in a microcentrifuge for 20-30 seconds and remove supernatant.
      2) Use a toothpick and vortexer to resuspend the cells in 100 μl(0.1mL) of STET buffer(8% sucrose; 5%Triton X-100;50 mM EDTA; Tris-Cl, pH 8.0) containing 1mg/mL lysozyme(prepare 1 mg/mLenzyme in STET just prior to use; 1mL= 10 preps).
      3)Place the suspension in a boiling water bath for 90 seconds.
      4) Spin in a microcentrifuge for 15 min at the highest speed.
      5)Remove the pellet (a gelatinous mass of cell debris) with a toothpick and discard.
      6) Precipitate nucleic acid at -20℃ for 30 minutes using an equal volume of isopropanol.
      7) Spin the tube in a microcentrifuge for 10 minutes at RTo to pellet the nucleic acids.
      8) Was pellet twice with 70% ethanol and dry thoroughly.
      9) Resuspend the pellet in 50 μL of sterile DI water or TE buffer.
    Spin Prep: We used the QIAprep Spin Miniprep Kit to run a spin prep and followed the instructions inside.

    (XX)Goal: Rerun the diagnostic gel stated yesterday (6/15/16)
    Experimental results:

    Good gel results, confirming that the PstI site has been removed in pSB416 GPD. pSB416 ready to be sequenced.


    Other activities:

    Incubated pSBIC3 mRPS12 TU and pSBIC3 mRPS12 mls (from last year iGEM team??) in LB-chloramphenicol broth @37C overnight at 250 rpm



6/17/16

(HC) Ran two digests for P413 GPD

      Analytical Digest:
        5 μL of DNA(Boiling Prep)
        1μL of PstI HF
        1μL of CutSmart buffer
        3μL of sterile DI water
      Preparative Digest:
        7μL of DNA(Spin Prep)
        1μL of NheI
        1μL of NsiI
        1μL of CutSmart buffer
    Gel Results:
      PREP GEL
        Lane 1: Ladder
        Lane 3: Preparative Digest
      INSERT NOTEBOOK PICTURE #001 HERE
    ANALYTICAL GEL
      Lane 1: Ladder
      Lane 2: Uncut (2μL of DNA and 8μL of water)
      Lane 3: Analytical Digest
    INSERT NOTEBOOK PICTURE #002 HERE

    (XX) Master plate results:
      Nothing grew on control plates (-His, -Ura, -Ura+Glycerol)
      Both parent and mutant grew on YPD and YPD+Paro, indicating that Paro has no effect on yeast growth
      Mutant cannot grow on YPG or YPG+Paro because of the mRPS12 gene knockout

    Other Activities:
      Cut p413GPD with NheI and NsiI to get rid of the illegal PstI site
      Massive gel to run boiling preps

    Conclusion:
      P413GPD cut looks like an uncut plasmid
      Massive gel has many blurry bands, which indicates that boiling preps are very impure


6/20/16

    (XX) Unltimate Goal:
      Put pSBIC3 mRPS12 TU into our own standardized yeast vectors
      Validate mls function by creating mls-yeGFP construct
    Goal for today:
      Digest and Harvest mRPS12 TU from pSBIC3(tube 1)
          5μL of DNA
          1μL of EcoRI-HF
          1μL of pstI-HF
          1μL of CutSmart buffer
          2μL of sterile water
        Digest and Harvest mRPS12 mls from pSBIC3 (tube 2)
          5μL of DNA
          1μL of EarI
          1μL of SpeI
          1μL of CutSmart buffer
          2μL of sterile water
        Gel Order: L, 1C, 1XX, 1XL, 2C, 2XX, 2XL
        **C-uncut plasmid, XX-Xintong, XL-Xander
        Gel results:
          Uncut does not show up on the gel
          Not a very good results but we harvested the bands anyway
        Other activiites:
          Excise bands from the gel and perform gel extraction (QIA kit):
            Tube 1 = 0.26 g
            Tube 2 = 0.14 g
            Tube 3 = 0.17 g
            *tube 3 contains p413 GPD with NsII/NheI cut
          Prepare Yeast Competent Cells
            10 mL YPD, inoculated at 250RPM @ 30C
            Both BY4741 and BY4741 mRPS12 is prepared

6/21/16

    (XX) Goal for Today:
      DNA Assembly
    Major Steps:
      We used nanodrop to determine the amount of DNA in tube 1 (TU) and tube 2 (mls)
      Tube 1: 4.5 ng/
      Tube 2: 4.0 ng/
      * Nano drop results indicate that we need to precipitate DNA for further experiment

      After precipitation, nanodrop reports 12.6 ng/ DNA

      NEBuilder HiFi DNA Assembly Cloning Kit:
        2μL Ear/SpeI digest pSBIC3 mls
        8μL fragment 16-4 (yeGFP)
        10μL HiFi Master
        *All three above, set the reaction on ice; 1:2 vecotr: insert
        Thermocycler
    Other Activities:
      Yeast competent cell from yesterday did not grow
      Prepare new yeast competent cells: 5 mL YPD in round bottom tubes

6/22/16

    (XX)Goal:
      Prepare Yeast Competent cells
      Diagnostic gel on mRPS12 TU minipreps
    Major Steps:
      Yeast competent cells:
        Spectromphotometer OD 260:
          1000 YPD Blank
          1000 KnockOut(KO) has 1.777 Abs.
          1000 Parent has 2.222 Abs.
        Regrow these cells into 15 mL broth for about 2 hrs:
          Take 0.5 mL current culture into 14.5 mL YPD
          Spectrophotometer Results:
            KO: 0.390 Abs.
            Parent: 0.692 Abs.
          Follow yeast competent cell protocol
        Diagnostic Gel:
          1: pSBIC3 mRPS12 TU miniprep 1 cut with EcoRI and PstI
          2: pSBIC3 mRPS12 TU miniprep 2 cut with EcoRI and PstI
          3: pSBIC3 mRPS12 TU miniprep 1 cut with PstI
          4: pSBIC3 mRPS12 TU miniprep 2 cut with PstI
          Gel Order: L, 1, 2, 3, 4
      Gel Results:
        For #2, fragment should be around 500bp
        Gel results indicate that we should redo minipreps or bad enzymes

6/23/16

    (XX) Goal:
      Test if the enzymes are still okay to work with
      Redo miniprep at the same time
    Major steps:
      Enzyme test:
        Tube 1: EcoRI-HF
        Tube 2: PstI-HF
        Tube 3: SpeI-HF
        Tube 4: EarI
      Gel Order: 1, 2, 3, 4, L
    Gel Results: enzymes are working properly

6/24/16

    (HC) After extracting P413 GPD fragment from the gel, I precipitated the DNA
        Precipitating DNA:
          1.)Add 1-2 μL of 5M NaCl to 30 μL of DNA
          2.)Add 62 μL of ice cold EtOH
          3.)Let sit on ice for 30 minutes
          4.)Centrifuge @ 0℃ for 10 minutes at 13 krpm
          5.)Remove supernatent
          6.)Fill tube to halfway point with 70% EtOH
          7.)Centrifuge @ 4℃ for 2 minutes
          8.)Remove supernant
          9.)Let dry
          10.)Add 6μL of Sterile Water.
        Obtained concentration of DNA with NanoDropper
      Nucleic Acid Concentratione A @ 260 nm A @ 280 nm 260/280 260/230
      75.7 ng/μL 1.514 0.922 1.64 .033
    (JM/CH)
    Today we are starting the day off by running a gel.
    We just called iGEM to confirm that we can indeed submit vectors as parts
    The gel we are running today is to confirm that we only have 1 PstI site in all of our plasmids
    The hope: to see one solid band from all of our plasmids except for the controls, the controls should be two bands because they still have two PstI sites
    Another thing we are doing today is we are making media LB Chloro.
    Our Gel:
1 2 3 4 5 6
416 control 426 control 416 CYC1 416 CYC1 426 GPD 426 GPD
    (XX) Talked about light switch design
      LexA promoter instread of Gal promoter
      PhyB/PIF3 red light system

6/25/16

    (XX) Miniprep mRPS12 TU and mRSP12 mls(total of 8 minipreps)

6/27/16

    (XX) Goal: Redo digest and harvest TU and mls
    Major steps:
      Cut mRPS12 TU with EcoRI/PstI
      Cut Mrps12 mls with EarI/SpeI
    Gel order: L,TU1a, TU1b, TU2a, TU2b, mls1a, mls1b, mls2a, mls2b
    Gel results:
      harvested TU bands from TU2a and TU2b (2 bands per tube)
        TU gel extract = 0.363 g
      harvested four mls bands (2 bands per tube)
        mls gel extract A = 0.342 g
        mls gel extract B = 0.255 g
    Other activities:
      QIA gel extraction kit
      Used nanodrop to test DNA amount:
        TU = 3.1 ng/
          mls A = 41.2 ng/
            mls B = 27.7 ng/
            *according to nanodrop readings, TU needs to be precipitate while we run NEBuilder on mls A and mls B
        (HC) Ran NEBuilder on P413 GPD and 16-1 fragment. 1μL DNA 3μL fragment 10μL NEBuilder HiFi DNA Assembily Master Mix 6μL Sterile DI water (JM/CH) The first thing we are doing is running the blunt end ligation protocol using NEB Quick Blunting Kit and the NEB Quick Ligation Kit This was done with 3 tubes of 416 CYC1 and 3 tubes of 426 GPD Next we did a transformation on one of the 416 CYC1 and one of the 426 GPD Those are spread on plates and are now incubating so we can use them tomorrow