Team:Duke/Notebook/July

From 2014.igem.org

(Difference between revisions)
Line 278: Line 278:
<div class="obj">Objective: Obtain backbones and switch parts into new backbones</div>
<div class="obj">Objective: Obtain backbones and switch parts into new backbones</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Gel cleanup of 3 backbones with Zymoclean prep kit</p>
 +
<ul>
 +
<li>Some tubes had to be divided into two samples for cleanup </li>
 +
<li> Final elution was 20 uL each into 2-3 tubes per backbone</li>
 +
<li> Nanodrops looked unclean: Most had high peak around 220-240 nm</li>
 +
<ul>
 +
<li> pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL </li>
 +
<ul><li> Peak ~220, then hump ~260</li></ul>
 +
<li> pSB6A1: 270 mg gel = 220 ng/uL</li>
 +
<ul><li> Peak, then hump as before</li></ul>
 +
<li> pSB3K3: 320 mg gel = 53.9 ng/uL</li>
 +
<ul><li> No clear sign of DNA in graph</li></ul>
 +
<li> pSB3K3: 230 mg gel = 20 ng/uL</li>
 +
<ul><li> No DNA</li></ul>
 +
<li> pSB3K3: 130 mg gel = 72 ng/uL</li>
 +
<ul><li> Possibly DNA, best option but may not be useful</li></ul>
 +
<li> pSB4K5: 290 mg gel = 43.4 ng/uL</li>
 +
<ul><li> No clear sign of DNA in graph</li></ul>
 +
<li> pSB4K5: 320 mg gel = 117.3 ng/uL</li>
 +
<ul><li> Possibly DNA, best option but may not be useful</li></ul>
 +
</ul>
 +
</ul>
 +
<p> Culture 3 colonies of pSB1C3-K608012</p>
 +
<ul><li> In order to switch into pSB6A1</li></ul>
</div>
</div>
<div class="obj">Objective: Insert crRNAs into pdCas9</div>
<div class="obj">Objective: Insert crRNAs into pdCas9</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Analytical Digest of promising crRNA inserts </p>
 +
<ul>
 +
<li> XbaI/BsaI digest</li>
 +
<li> In order to confirm that plasmids are not uncut pdCas9</li>
 +
<li> Samples 7-1-1,2,3 and 7-2-7,8,9 (6 tubes) plus pdCas9 as control</li>
 +
</ul>
 +
<p> Agarose gel of digest results:</p>
 +
<ul>
 +
<li> 1. pdCas9</li>
 +
<li> 2-4. pdCas9-crRNA-GFP1, samples 1,2,3</li>
 +
<li> 5-7. pdCas9-crRNA-GFP2, samples 7,8,9</li>
 +
<li> Expected: </li>
 +
<ul>
 +
<li> 2 bands at 4.2 and 5.1 kb in pdCas9</li>
 +
<li> 1 band at 9.3 kb in successful plasmids with inserts</li>
 +
</ul>
 +
<li> Results: </li>
 +
<ul>
 +
<li> pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band</li>
 +
<li> Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.</li>
 +
</ul>
 +
</ul>
</div>
</div>
<div class="obj">Objective: Make pDGC3</div>
<div class="obj">Objective: Make pDGC3</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Culture 3 colonies each from pSB1C3-R0011 and pSB1C3-I13500</p>
</div>
</div>
<div class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</div>
<div class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</div>
 +
<div class="ppl">Mitch and Sargis</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Culture pSB1C3-I13521 (pTet-RFP)</p>
 +
<ul>
 +
<li> For flow </li>
 +
<li> In aTc and non-aTc </li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 298: Line 347:
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Miniprep 3 copies of pSB1C3-K608012</p>
 +
<ul><li> concentrations 361, 324, 362 ng/uL</li></ul>
 +
<p> Prep-scale digest of pSB1C3-K608012</p>
 +
<ul>
 +
<li> 2 miniprep tubes, 50 uL DNA in 100 uL each</li>
 +
<li> With EcoRI and SpeI</li>
 +
<li> 2+ hrs at 37C</li>
 +
</ul>
 +
<p> Gel extraction and cleanup to isolate K608012</p>
 +
<ul>
 +
<li> Cut lower band</li>
 +
<li> Zymoclean prep kit</li>
 +
<li> Each of 2 bands divided into two tubes during cleanup</li>
 +
<ul>
 +
<li> 1A: 280 mg = </li>
 +
<li> 1B: 240 mg = </li>
 +
<li> 2A: 200 mg = </li>
 +
<li> 2B: 200 mg = 62.5 ng/uL (20 uL)</li>
 +
</ul>
 +
<li> Graphs on nanodrop did not look ideal: peak lower than usual</li>
 +
</ul>
</div>
</div>
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block</div>
<div class="obj">Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Miniprep pSB1C3-R0011 and pSB1C3-I13500</p>
 +
<ul>
 +
<li> 3 copies each</li>
 +
<li> Concentrations</li>
 +
<ul>
 +
<li> pSB1C3-R0011: 181, 229, 194 ng/uL</li>
 +
<li> pSB1C3-I13500: 321.5, 228, 300.4 ng/uL</li>
 +
</ul>
 +
</ul>
 +
<p> Prep scale digests of two tubes from each miniprep</p>
 +
<ul>
 +
<li> pSB1C3-R0011 with SpeI/PstI</li>
 +
<ul> <li> no extraction necessary</li></ul>
 +
<li> pSB1C3-I13521 with XbaI/PstI</li>
 +
<ul> <li> to extract I13521 insert</li></ul>
 +
<li> 100 uL total reaction per tube</li>
 +
<li> 2-3 hrs at 37C</li>
 +
</ul>
 +
<p> Agarose gel of pSB1C3-I13500 XbaI/PstI digests</p>
 +
<ul>
 +
<li> Intended for gel extraction</li>
 +
<li> Expected 2 bands, but only one present</li>
 +
<li> No extraction performed</li>
 +
</ul>
 +
<p> Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests</p>
 +
<ul>
 +
<li> Single band appears correct</li>
 +
<li> see below for gel picture</li>
 +
</ul>
 +
<p> PCR cleanup of pSB1C3-R0011 SpeI/PstI digests</p>
 +
<ul>
 +
<li> concentrations 124.8, 157.3 ng/uL (30 uL each)</li>
 +
</ul>
</div>
</div>
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
<div class="lab">
<div class="lab">
 +
<p> Miniprep pdCas9</p>
 +
<ul>
 +
<li> concentration 167.4 ng/uL</li>
 +
</ul>
 +
<p> Prep-scale digest of pSB1C3-R0011</p>
 +
<ul>
 +
<li> 1 miniprep with XbaI/PstI</li>
 +
<li> To extract pSB1C3 backbone</li>
 +
</ul>
 +
<p> Agarose gel of pSB1C3-R0011 XbaI/PstI digest</p>
 +
<ul>
 +
<li> Intended for gel extraction</li>
 +
<li> Expected 2 bands, but only one present</li>
 +
<li> No extraction performed</li>
 +
</ul>
 +
<p> PCR of dCas9-tracrRNA and anti-tracrRNA</p>
 +
<ul>
 +
<li> 4 tubes each, with pdCas9 as template in 0, 0.4, 0.7, and 1.0 uL quantities</li>
 +
<li> oligos dCas9tracr-up and dCas9tracr-dn for dCas9-tracrRNA PCR</li>
 +
<li> oligos dCas9tracr-up and tracrRNA-dn for anti-tracrRNA PCR</li>
 +
<li> Q5 polymerase, with 64C annealing and 2 min extension</li>
 +
</ul>
 +
<p> Agarose gel of PCR (and digest) results:</p>
 +
<ul>
 +
<li> Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)</li>
 +
<li> Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)</li>
 +
<li> Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests</li>
 +
<li> Results: </li>
 +
<ul>
 +
<li> dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes</li>
 +
<li> tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension. </li>
 +
<ul>
 +
<li> tracrRNA PCR should be done with shorter extension time</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<p> PCR cleanup of dCas9-tracrRNA PCR</p>
 +
<ul>
 +
<li> Concentration >200 ng/uL</li>
 +
</ul>
 +
</div>
-
</div>
 
<div class="obj">Objective: Test BsaI and XbaI enzymes</div>
<div class="obj">Objective: Test BsaI and XbaI enzymes</div>
<div class="lab">
<div class="lab">
-
 
+
<p> Analytical digest of dCas9</p>
 +
<ul>
 +
<li> One tube with BsaI/EcoRI</li>
 +
<li> One tube with XbaI/EcoRI</li>
 +
<ul>
 +
<li> XbaI from tube with exp. 5/14</li>
 +
</ul>
 +
<li> One tube with XbaI/EcoRI</li>
 +
<ul>
 +
<li> XbaI from tube with exp. 2/15</li>
 +
</ul>
 +
</ul>
 +
<p> Agarose gel of digest:</p>
 +
<ul>
 +
<li> 1. BsaI/EcoRI: expected 3 bands at 2.7, 2.9, and 3.5 kb</li>
 +
<li> 2. XbaI (5/14) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb</li>
 +
<li> 3. XbaI (2/15) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb</li>
 +
<li> Results: Lanes 1 and 3 look as expected. Lane two has one extra band at 3.5 kb corresponding to partial digestion with XbaI. 5/14 XbaI tube is ineffective and has been thrown out</li>
 +
</ul>
</div>
</div>
</div>
</div>

Revision as of 13:57, 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

Gel cleanup of 3 backbones with Zymoclean prep kit

  • Some tubes had to be divided into two samples for cleanup
  • Final elution was 20 uL each into 2-3 tubes per backbone
  • Nanodrops looked unclean: Most had high peak around 220-240 nm
    • pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL
      • Peak ~220, then hump ~260
    • pSB6A1: 270 mg gel = 220 ng/uL
      • Peak, then hump as before
    • pSB3K3: 320 mg gel = 53.9 ng/uL
      • No clear sign of DNA in graph
    • pSB3K3: 230 mg gel = 20 ng/uL
      • No DNA
    • pSB3K3: 130 mg gel = 72 ng/uL
      • Possibly DNA, best option but may not be useful
    • pSB4K5: 290 mg gel = 43.4 ng/uL
      • No clear sign of DNA in graph
    • pSB4K5: 320 mg gel = 117.3 ng/uL
      • Possibly DNA, best option but may not be useful

Culture 3 colonies of pSB1C3-K608012

  • In order to switch into pSB6A1
Objective: Insert crRNAs into pdCas9

Analytical Digest of promising crRNA inserts

  • XbaI/BsaI digest
  • In order to confirm that plasmids are not uncut pdCas9
  • Samples 7-1-1,2,3 and 7-2-7,8,9 (6 tubes) plus pdCas9 as control

Agarose gel of digest results:

  • 1. pdCas9
  • 2-4. pdCas9-crRNA-GFP1, samples 1,2,3
  • 5-7. pdCas9-crRNA-GFP2, samples 7,8,9
  • Expected:
    • 2 bands at 4.2 and 5.1 kb in pdCas9
    • 1 band at 9.3 kb in successful plasmids with inserts
  • Results:
    • pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band
    • Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.
Objective: Make pDGC3

Culture 3 colonies each from pSB1C3-R0011 and pSB1C3-I13500

Objective: Test effectiveness of DH5alpha-Z1 strain
Mitch and Sargis

Culture pSB1C3-I13521 (pTet-RFP)

  • For flow
  • In aTc and non-aTc

July 11
Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of pSB1C3-K608012

  • concentrations 361, 324, 362 ng/uL

Prep-scale digest of pSB1C3-K608012

  • 2 miniprep tubes, 50 uL DNA in 100 uL each
  • With EcoRI and SpeI
  • 2+ hrs at 37C

Gel extraction and cleanup to isolate K608012

  • Cut lower band
  • Zymoclean prep kit
  • Each of 2 bands divided into two tubes during cleanup
    • 1A: 280 mg =
    • 1B: 240 mg =
    • 2A: 200 mg =
    • 2B: 200 mg = 62.5 ng/uL (20 uL)
  • Graphs on nanodrop did not look ideal: peak lower than usual
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Miniprep pSB1C3-R0011 and pSB1C3-I13500

  • 3 copies each
  • Concentrations
    • pSB1C3-R0011: 181, 229, 194 ng/uL
    • pSB1C3-I13500: 321.5, 228, 300.4 ng/uL

Prep scale digests of two tubes from each miniprep

  • pSB1C3-R0011 with SpeI/PstI
    • no extraction necessary
  • pSB1C3-I13521 with XbaI/PstI
    • to extract I13521 insert
  • 100 uL total reaction per tube
  • 2-3 hrs at 37C

Agarose gel of pSB1C3-I13500 XbaI/PstI digests

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests

  • Single band appears correct
  • see below for gel picture

PCR cleanup of pSB1C3-R0011 SpeI/PstI digests

  • concentrations 124.8, 157.3 ng/uL (30 uL each)
Objective: Switch dCas9-tracrRNA into pSB1C3

Miniprep pdCas9

  • concentration 167.4 ng/uL

Prep-scale digest of pSB1C3-R0011

  • 1 miniprep with XbaI/PstI
  • To extract pSB1C3 backbone

Agarose gel of pSB1C3-R0011 XbaI/PstI digest

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

PCR of dCas9-tracrRNA and anti-tracrRNA

  • 4 tubes each, with pdCas9 as template in 0, 0.4, 0.7, and 1.0 uL quantities
  • oligos dCas9tracr-up and dCas9tracr-dn for dCas9-tracrRNA PCR
  • oligos dCas9tracr-up and tracrRNA-dn for anti-tracrRNA PCR
  • Q5 polymerase, with 64C annealing and 2 min extension

Agarose gel of PCR (and digest) results:

  • Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests
  • Results:
    • dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes
    • tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension.
      • tracrRNA PCR should be done with shorter extension time

PCR cleanup of dCas9-tracrRNA PCR

  • Concentration >200 ng/uL
Objective: Test BsaI and XbaI enzymes

Analytical digest of dCas9

  • One tube with BsaI/EcoRI
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 5/14
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 2/15

Agarose gel of digest:

  • 1. BsaI/EcoRI: expected 3 bands at 2.7, 2.9, and 3.5 kb
  • 2. XbaI (5/14) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • 3. XbaI (2/15) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • Results: Lanes 1 and 3 look as expected. Lane two has one extra band at 3.5 kb corresponding to partial digestion with XbaI. 5/14 XbaI tube is ineffective and has been thrown out

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"