Team:Duke/Notebook/July

From 2014.igem.org

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</ul>
</ul>
<li>Results:</li>
<li>Results:</li>
 +
<ul>
 +
<li>Gel 1 appears to only be recircularized </li>
 +
<li> Gel 2 has some promising inserts:</li>
 +
<ul>
 +
<li> GFP1 samples 1,2, and 3 from 7/7 transformation</li>
 +
<li> GFP2 samples 7,8, and 9 from 7/7 transformation</li>
 +
</ul>
 +
</ul>
 +
<li> Glycerol stocks frozen for these six samples</li>
 +
<li>Next Steps: </li>
 +
<ul>
 +
<li> Run digest with BsaI to confirm that plasmids are not uncut pdCas9</li>
 +
<li> Sequence to confirm presence of insert (using oligos ordered 7/8/14)</li>
 +
</ul>
</ul>
</ul>
-
</div>
 
</div>
</div>
 +
<div class="obj">Objective: Obtain new backbones and switch inserts into new backbones</div>
 +
<div class="lab">
 +
<p>Miniprep 3 new backbones </p>
 +
<ul>
 +
<li>pSB6A1-J04450 (3 copies)</li>
 +
<ul>
 +
<li> concentrations 437.6, 483.9, and 450.1 ng/uL</li>
 +
</ul>
 +
<li>pSB3K3-I20260 (3 copies)</li>
 +
<ul>
 +
<li> concentrations 114.6, 120.0, and 97.3 ng/uL</li>
 +
</ul>
 +
<li>pSB4K5-J04450 (3 copies)</li>
 +
<ul>
 +
<li> concentrations 129.3, 165.9, and 155.1 ng/uL</li>
 +
</ul>
 +
</ul>
 +
<p>Prep-scale digest of all 9 backbone tubes</p>
 +
<ul>
 +
<li> 50 uL DNA in 60 uL total reaction</li>
 +
<li>EcoRI-HF/Spe-HF in cutsmart </li>
 +
<li> 3 hours at 37C</li>
 +
</ul>
 +
<p> Agarose gel extraction of 3 backbones</p>
 +
<ul>
 +
<li> Gel 1, Left: pSB6A1</li>
 +
<li> Gel 1, Right: pSB3K3</li>
 +
<li> Gel 2, Left: pSB4K5</li>
 +
<li> All 3 lanes had 2 bands as expected</li>
 +
<ul><li> upper band (backbone) in 3K3 had messy trail behind it (not extracted)</li></ul>
 +
<li> All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well</li>
 +
<li> top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C</li>
 +
</ul>
 +
<p> Streak plate from frozen stock</p>
 +
<ul>
 +
<li> pSB1C3-K608012</li>
 +
<li> In order to switch into pSB6A1</li>
 +
</ul>
 +
</div>
 +
<div class="obj">Objective: Remake new version of pDGC3</div>
 +
<div class="lab">
 +
<p>Transformation from iGEM distribution kit plates </p>
 +
<ul>
 +
<li>Plate 2-6D: pSB1C3-BBa_R0011 </li>
 +
<ul><li> Engineered Lac promoter (not affected by glucose)</li></ul>
 +
<li> Plate 3-15O: pSB1C3-BBa_I13500</li>
 +
<ul> <li> Strong RBS-GFP</li> </ul>
 +
<li> Plated on LB+Cm</li>
 +
</ul>
 +
</div>
 +
</div>
<div class="day">
<div class="day">

Revision as of 12:49, 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

June 1
Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

  • Followed Charlie’s Cloning Protocols
    • Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
  • Plated the transformed DNA and put in incubator at 37C
  • Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

  • Look at plates, compare cultures

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