Team:Duke/Notebook/July

From 2014.igem.org

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<a id="jun1"><h2>June 1</h2></a>
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<a id="july10"><h2>July 10</h2></a>
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<div class="obj">Objective: Transform Chemically competent E. Coli (CCEC)</div>
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<div class="obj">Objective: Obtain backbones and switch parts into new backbones</div>
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<div class="ppl">Matthew Faw</div>
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<div class="lab">
<div class="lab">
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<p> Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA</p>
 
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<ul>
 
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<li>Followed Charlie’s Cloning Protocols</li>
 
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<ul>
 
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<li>Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful</li>
 
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</ul>
 
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<li>Plated the transformed DNA and put in incubator at 37C</li>
 
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<li>Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw</li>
 
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</ul>
 
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<p> Next Steps:</p>
 
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<ul>
 
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<li>Look at plates, compare cultures</li>
 
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</ul>
 
</div>
</div>
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<div class="obj">Objective: Insert crRNAs into pdCas9</div>
 +
<div class="lab">
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</div>
</div>
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<div class="obj">Objective: Make pDGC3</div>
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<div class="lab">
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</div>
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<div class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</div>
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<div class="lab">
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</div>
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</div>
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<div class="day">
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<a id="july11"><h2>July 11</h2></a>
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<div class="obj">Objective: Switch K608012 into pSB6A1</div>
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<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block</div>
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<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Test BsaI and XbaI enzymes</div>
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<div class="lab">
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</div>
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</div>
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<div class="day">
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<a id="july14"><h2>July 14</h2></a>
 +
<div class="obj">Objective: insert crRNAs into pdCas9</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Create pTet-anti-tracrRNA</div>
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<div class="lab">
 +
 +
</div>
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</div>
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<div class="day">
 +
<a id="july15"><h2>July 15</h2></a>
 +
<div class="obj">Objective: Create pTet-anti-tracrRNA</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
 +
<div class="lab">
 +
 +
</div>
 +
</div>
 +
 +
 +
<div class="day">
 +
<a id="july16"><h2>July 16</h2></a>
 +
<div class="obj">Objective: Create pTet-anti-tracrRNA</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
 +
<div class="lab">
 +
 +
</div>
 +
<div class="obj">Objective: Insert crRNAs into pdCas9</div>
 +
<div class="lab">
 +
 +
</div>
 +
</div>
<div class="day">
<div class="day">

Revision as of 13:07, 15 August 2014

May 2014
Month 2 of Project
sun mon tue wed thu fri sat
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

June 2014
Month 3 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30

July 9
Objective: Insert crRNAs into pdCas9

Miniprep 40 cultures of potential crRNA inserts in pdCas9

  • Labeled with three numbers
    • Date of transformation - crRNA-GFPx - sample number

700uL of each culture saved to freeze in glycerol if colonies appear successful

  • Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

  • All 40 tubes + 2 pdCas9 tubes as control
  • 1 uL DNA in 10 uL digest for 2 hrs at 37C
  • SacI-HF and NheI-HF in cutsmart

Made and ran 1.5% agarose/TBE gels

  • To differentiate between small band differences
  • Gel 1:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Gel 2:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Expected results:
    • pdCas9 and successful inserts:
      • 3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band
    • unsuccessful recircularizations:
      • 3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band
  • Results:
    • Gel 1 appears to only be recircularized
    • Gel 2 has some promising inserts:
      • GFP1 samples 1,2, and 3 from 7/7 transformation
      • GFP2 samples 7,8, and 9 from 7/7 transformation
  • Glycerol stocks frozen for these six samples
  • Next Steps:
    • Run digest with BsaI to confirm that plasmids are not uncut pdCas9
    • Sequence to confirm presence of insert (using oligos ordered 7/8/14)
Objective: Obtain new backbones and switch inserts into new backbones

Miniprep 3 new backbones

  • pSB6A1-J04450 (3 copies)
    • concentrations 437.6, 483.9, and 450.1 ng/uL
  • pSB3K3-I20260 (3 copies)
    • concentrations 114.6, 120.0, and 97.3 ng/uL
  • pSB4K5-J04450 (3 copies)
    • concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

  • 50 uL DNA in 60 uL total reaction
  • EcoRI-HF/Spe-HF in cutsmart
  • 3 hours at 37C

Agarose gel extraction of 3 backbones

  • Gel 1, Left: pSB6A1
  • Gel 1, Right: pSB3K3
  • Gel 2, Left: pSB4K5
  • All 3 lanes had 2 bands as expected
    • upper band (backbone) in 3K3 had messy trail behind it (not extracted)
  • All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
  • top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

  • pSB1C3-K608012
  • In order to switch into pSB6A1
Objective: Remake new version of pDGC3

Transformation from iGEM distribution kit plates

  • Plate 2-6D: pSB1C3-BBa_R0011
    • Engineered Lac promoter (not affected by glucose)
  • Plate 3-15O: pSB1C3-BBa_I13500
    • Strong RBS-GFP
  • Plated on LB+Cm

July 10
Objective: Obtain backbones and switch parts into new backbones
Objective: Insert crRNAs into pdCas9
Objective: Make pDGC3
Objective: Test effectiveness of DH5alpha-Z1 strain

July 11
Objective: Switch K608012 into pSB6A1
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block
Objective: Switch dCas9-tracrRNA into pSB1C3
Objective: Test BsaI and XbaI enzymes

July 14
Objective: insert crRNAs into pdCas9
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block
Objective: Switch K608012 into pSB6A1
Objective: Switch dCas9-tracrRNA into pSB1C3
Objective: Create pTet-anti-tracrRNA

July 15
Objective: Create pTet-anti-tracrRNA
Objective: Switch dCas9-tracrRNA into pSB1C3
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block
Objective: Switch K608012 into pSB6A1

July 16
Objective: Create pTet-anti-tracrRNA
Objective: Switch dCas9-tracrRNA into pSB1C3
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block
Objective: Switch K608012 into pSB6A1
Objective: Insert crRNAs into pdCas9

Retrieved from "http://2014.igem.org/Team:Duke/Notebook/July"