Difference between revisions of "Team:RHIT/Notebook"

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   <ul> Massive gel has many blurry bands, which indicates that boiling preps are very impure </ul>
 
   <ul> Massive gel has many blurry bands, which indicates that boiling preps are very impure </ul>
 
</ul> <br/><br/>
 
</ul> <br/><br/>
 +
 +
<h2> 6/20/16 </h2>
 +
<ul>(XX) Unltimate Goal:</ul>
 +
<ul>
 +
  <ul>Put pSBIC3 mRPS12 TU into our own standardized yeast vectors</ul>
 +
  <ul>Validate mls function by creating mls-yeGFP construct</ul>
 +
</ul>
 +
<ul>Goal for today:</ul>
 +
<ul>
 +
  <ul>Digest and Harvest mRPS12 TU from pSBIC3(tube 1)
 +
<ul>
 +
      <ul>5μL of DNA</ul>
 +
      <ul>1μL of EcoRI-HF </ul>
 +
      <ul>1μL of pstI-HF</ul>
 +
      <ul>1μL of CutSmart buffer</ul>
 +
      <ul>2μL of sterile water</ul>
 +
</ul>
 +
 +
  <ul>Digest and Harvest mRPS12 mls from pSBIC3 (tube 2)</ul>
 +
      <ul><ul>5μL of DNA</ul>
 +
      <ul>1μL of EarI </ul>
 +
      <ul>1μL of SpeI</ul>
 +
      <ul>1μL of CutSmart buffer</ul>
 +
      <ul>2μL of sterile water</ul>
 +
</ul>
 +
  <ul>Gel Order: L, 1C, 1XX, 1XL, 2C, 2XX, 2XL</ul>
 +
  <ul>**C-uncut plasmid, XX-Xintong, XL-Xander</ul>
 +
  <ul>Gel results:</ul>
 +
<ul>
 +
    <ul>Uncut does not show up on the gel</ul>
 +
    <ul>Not a very good results but we harvested the bands anyway</ul>
 +
</ul>
 +
  <ul>Other activiites:</ul>
 +
<ul>
 +
    <ul>Excise bands from the gel and perform gel extraction (QIA kit):</ul>
 +
<ul>
 +
        <ul>Tube 1 = 0.26 g</ul>
 +
        <ul>Tube 2 = 0.14 g</ul>
 +
        <ul>Tube 3 = 0.17 g</ul>
 +
        <ul>*tube 3 contains p413 GPD with NsII/NheI cut</ul>
 +
</ul>
 +
    <ul> Prepare Yeast Competent Cells</ul>
 +
<ul>
 +
        <ul>10 mL YPD, inoculated at 250RPM @ 30C</ul>
 +
        <ul>Both BY4741 and BY4741 mRPS12 is prepared</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
<br>
 +
 +
<h2>6/21/16</h2>
 +
 +
  
  
 
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Revision as of 14:45, 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
    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
    <u1> ANALYTICAL GEL
      Lane 1: Ladder
      Lane 2: Uncut (2μL of DNA and 8μL of water)
      Lane 3: Analytical Digest
    INSERT NOTEBOOK PICTURE #002 HERE

</ul>

    (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



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