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

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(Autocreate 2012/12/05 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes)
Current revision (09:46, 8 December 2012) (view source)
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==Entry title==
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==December 5, 2012==
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* Insert content here...
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'''Results from last 11/23/12 ligation'''
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* None of the colonies on any plates grew
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* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
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----
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* Repeat PCR of H2B and LOV inserts with new primers
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* Note: Primers add a XbaI site upstream and the SpeI site downstream
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* 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 / LOV / H2B +His tag/ pSB1A3 right end
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*For H2B-LOV construct
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# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
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# LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
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*For LOV-H2B construct
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# H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
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# LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev
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Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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| Reagents || H2B || LOV
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|-
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| Plasmid DNA || 0.5 μL || 0.5
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|-
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| primer 1 (10 μM) || 1.0 || 1.0
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|-
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| primer 2 (10 μM) || 1.0 || 1.0
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|-
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| 2x GoTaq mix || 12.5 || 12.5
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|-
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| dH<sub>2</sub>O || 10.0 || 10.0
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|-
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| Total || 25.0 || 25.0
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|}
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PCR program:
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* 95°C, 3 min.
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* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
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* 72°C, 3 min.
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* 4°C ∞
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* Clean up PCR products using the Zymo Clean and Concentrator kit
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* Be sure to elute using 15 μL dH<sub>2</sub>O
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{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
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|Construct || Product || 260 || 280 || 260/280 || ng/μL
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|-
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|H2B-LOV || H2B 1 || 0.061 || 0.033 || 1.863 || 60.545
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|-
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|H2B-LOV || H2B 2 || 0.056 || 0.03 || 1.857 || 56.22
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|-
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|H2B-LOV || LOV 1 || 0.063 || 0.033 || 1.903 || 62.673
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|-
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|H2B-LOV || LOV 2 || 0.06 || 0.04 || 1.85 || 59.844
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|-
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|LOV-H2B || H2B 1 || 0.072 || 0.033 || 1.807 || 72.381
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|-
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|LOV-H2B || H2B 2 || 0.06 || 0.036 || 1.799 || 59.925
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|-
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|LOV-H2B || LOV 1 || 0.006 || 0.036 || 1.849 || 65.881
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|-
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|LOV-H2B || LOV 2 || 0.065 || 0.035 || 1.839 || 65.285
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|}
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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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