Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21

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*'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
*'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
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'''Assemblies'''
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#
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'''Ligation'''
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* Use 20 ng of vector for each ligation = '''1 μL pSB1A3'''
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* Use 2x moles of H2B insert, relative to vector:  xμL  H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = '''1.28 μL H2B'''
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* Use 2x moles of LOV insert, relative to vector:  xμL  LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = '''0.8 μL LOV'''
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{| class="wikitable" width=400px
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|   || H2B || LOV  || Negative Control
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|-
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| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || 0
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|-
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| Vector DNA (50 ng) || 1.0  || 1.0 || 1.0
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|-
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| 2x Roche Rapid Ligation buffer || 7.0 || 7.0 || 7.0
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|-
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| New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0
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|-
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| dH<sub>2</sub>O || 4.0 || 4.0 || 5.0
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|-
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| TOTAL || 14.0 || 14.0 || 14.0
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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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Revision as of 16:59, 21 November 2012

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11/21/12

  • Repeat PCR of H2B and LOV inserts
  • Note: Primers add a XbaI site upstream and the SpeI, NotI, and PstI sites downstream
  1. H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
  2. LOV plasmid + BB_LOV fwd + BB_LOV rev
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 ∞
Expected:
1. H2B = 410
2. LOV = 461

Results of the digests are shown. Image:KAH_Fermentas_GeneRuler_1kbplus.jpg

Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.

  • ---Karmella 15:56, 21 November 2012 (EST): Digest, purification, and ligation

Assemblies

Ligation

  • Use 20 ng of vector for each ligation = 1 μL pSB1A3
  • Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = 1.28 μL H2B
  • Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = 0.8 μL LOV
  H2B LOV Negative Control
Insert DNA (2x mol vector) 1.0 μL 1.0 0
Vector DNA (50 ng) 1.0 1.0 1.0
2x Roche Rapid Ligation buffer 7.0 7.0 7.0
New England Biolabs T4 ligase 1.0 1.0 1.0
dH2O 4.0 4.0 5.0
TOTAL 14.0 14.0 14.0
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.

Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101




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