Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/05

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(11/21/12)
Current revision (09:46, 8 December 2012) (view source)
(December 5, 2012)
 
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==December 5, 2012==
==December 5, 2012==
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==11/21/12==
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'''Results from last 11/23/12 ligation'''
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'''Results from last week's ligation'''
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* None of the colonies on any plates grew
* None of the colonies on any plates grew
* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
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* Note: Primers add a XbaI site upstream and the SpeI site downstream
* Note: Primers add a XbaI site upstream and the SpeI site downstream
* Final Gibson assembly products
* Final Gibson assembly products
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# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
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# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
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# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
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# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
*For H2B-LOV construct
*For H2B-LOV construct
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* Clean up PCR products using the Zymo Clean and Concentrator kit
* Clean up PCR products using the Zymo Clean and Concentrator kit
* Be sure to elute using 15 μL dH<sub>2</sub>O
* Be sure to elute using 15 μL dH<sub>2</sub>O
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----
 
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*'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
 
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'''Assemblies'''
 
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# NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
 
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# NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
 
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# NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
 
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# Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
 
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# Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
 
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# Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
 
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* Digests:
 
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** In a 30 μL reaction, cut H2B with XbaI and SpeI
 
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** In a 30 μL reaction, cut LOV with XbaI and SpeI
 
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
|-
|-
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| Reagent || H2B PCR || LOV PCR
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|Construct || Product || 260 || 280 || 260/280 || ng/μL
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|-
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| DNA || 15.0 || 15.0
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|H2B-LOV || H2B 1 || 0.061 || 0.033 || 1.863 || 60.545
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|-
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| XbaI || 1.0|| 1.0
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|H2B-LOV || H2B 2 || 0.056 || 0.03 || 1.857 || 56.22
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|-
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| SpeI || 1.0 || 1.0
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|H2B-LOV || LOV 1 || 0.063 || 0.033 || 1.903 || 62.673
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|-
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| 10x buffer || 3.0 || 3.0
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|H2B-LOV || LOV 2 || 0.06 || 0.04 || 1.85 || 59.844
|-
|-
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| dH<sub>2</sub>O || 10.0 || 10.0
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|LOV-H2B || H2B 1 || 0.072 || 0.033 || 1.807 || 72.381
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|-
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| Total vol. || 30. 0 || 30.0  
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|LOV-H2B || H2B 2 || 0.06 || 0.036 || 1.799 || 59.925
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|}
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* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
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* Cut band out of gel (use UV box)
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Results from gel recovery:
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
|-
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| Sample|| 260|| 260/280 || ng/μL
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|LOV-H2B || LOV 1 || 0.006 || 0.036 || 1.849 || 65.881
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|-
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| H2B - X/S 1 || 0.016 ||1.807|| 15.961
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|LOV-H2B || LOV 2 || 0.065 || 0.035 || 1.839 || 65.285
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|-
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| H2B - X/S 2 || 0.009 || 1.976 || 8.619
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|-
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| LOV - X/S 1 || 0.006|| 1.694 || 6.455
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|-
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| LOV - X/S 2 || 0.025|| 1.883 || 25.363
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|}
|}
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[[Image:VN_Gel_11-21-12.png|Results of from the digests are shown.]]
 
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[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
 
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# H2B = 410
 
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# LOV = 461
 
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* There were two samples made of each PCR reaction.  The two from the H2B are shown on the left, and the two from the LOV are shown on the right.
 
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'''Dephosphorylation''' (11/23/12)
 
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> Dephosphorylation (Roche)
 
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# pSB1A3 - X/S = 2000 bp
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
 
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|-
 
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| <u>Reagent</u> || <u>Volume</u>
 
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|-
 
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| DNA (clean digest) || 11.0 (~200 ng)
 
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|-
 
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| 10x phos. buffer || 1.5
 
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|-
 
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| phosphatase || 0.5
 
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|-
 
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| dH<sub>2</sub>O || 2.0
 
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|-
 
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| &nbsp; || 15 μL
 
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|}
 
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* Incubate at 37°C/ 10 min.
 
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* Heat inactivate at 75°C/ 2 min.
 
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* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
 
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'''Ligation'''
 
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* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
 
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* Use 3x moles of H2B insert, relative to vector:  xμL  H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least '''0.8 μL H2B'''
 
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* Use 3x moles of LOV insert, relative to vector:  xμL  LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least '''0.5 μL LOV'''
 
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{| class="wikitable" width=700px
 
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| &nbsp; || H2B || LOV  || Negative Control || H2B || LOV  || Negative Control
 
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|-
 
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| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || ---
 
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|-
 
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| Vector DNA (50 ng) || 1.5  || 1.5 || 1.5 || 1.5  || 1.5 || 1.5
 
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|-
 
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| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0
 
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|-
 
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| T4 ligase || 1.0  NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche
 
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|-
 
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| dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5
 
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|-
 
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| TOTAL || 10.0 || 10.0 || 10.0  || 10.0 || 10.0 || 10.0
 
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|-
 
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
 
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|}
 
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
 
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Current revision

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December 5, 2012

Results from last 11/23/12 ligation

  • None of the colonies on any plates grew
  • Will retry cloning for each (H2B, LOV) by using the Gibson assembly method

  • Repeat PCR of H2B and LOV inserts with new primers
  • Note: Primers add a XbaI site upstream and the SpeI site downstream
  • Final Gibson assembly products
  1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
  • For H2B-LOV construct
  1. H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
  2. LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
  • For LOV-H2B construct
  1. H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
  2. LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev

Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct

Reagents H2B LOV
Plasmid DNA 0.5 μL 0.5
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 12.5 12.5
dH2O 10.0 10.0
Total 25.0 25.0

PCR program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
  • 72°C, 3 min.
  • 4°C ∞
  • Clean up PCR products using the Zymo Clean and Concentrator kit
  • Be sure to elute using 15 μL dH2O
Construct Product 260 280 260/280 ng/μL
H2B-LOV H2B 1 0.061 0.033 1.863 60.545
H2B-LOV H2B 2 0.056 0.03 1.857 56.22
H2B-LOV LOV 1 0.063 0.033 1.903 62.673
H2B-LOV LOV 2 0.06 0.04 1.85 59.844
LOV-H2B H2B 1 0.072 0.033 1.807 72.381
LOV-H2B H2B 2 0.06 0.036 1.799 59.925
LOV-H2B LOV 1 0.006 0.036 1.849 65.881
LOV-H2B LOV 2 0.065 0.035 1.839 65.285


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