Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06

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* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
-
 
+
'''Ligation'''
 +
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
 +
* Use 1x moles of H2B insert, relative to vector:  1xμL  H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least '''0.07 μL H2B'''
 +
* Use 1x moles of LOV insert, relative to vector:  1xμL  LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least '''0.07 μL LOV'''
 +
* Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity
{| class="wikitable" width=700px
{| class="wikitable" width=700px
-
|   || H2B || LOV || Negative Control || H2B || LOV || Negative Control
+
|   || H2B-LOV || LOV-H2B  || Negative Control  
|-
|-
-
| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || ---
+
| H2B-LOV H2B insert DNA (1x mol vector) || 1.0 μL || 0 || 0  
|-
|-
-
| Vector DNA (50 ng) || 1.5  || 1.5 || 1.5 || 1.5  || 1.5 || 1.5
+
| H2B-LOV LOV insert DNA (1x mol vector) || 1.0 μL || 0 || 0
|-
|-
-
| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0
+
| LOV-H2B H2B insert DNA (1x mol vector) || 0 μL || 1.0 || 0
|-
|-
-
| T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche
+
| LOV-H2B LOV insert DNA (1x mol vector) || 0 μL || 1.0 || 0
-
|-
+
-
| dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5
+
|-
|-
-
| TOTAL || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0  
+
| Vector DNA (39 ng) || 3.0 || 3.0 || 3.0  
-
|-  
+
|-
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
+
| Gibson master mix || 15.0 || 15.0 || 15.0
 +
|-
 +
| Total volume || 20.0 || 20.0 || 18.0
 +
|-
 +
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.
|}
|}
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
 +
[[Image:VN_Gibson_Assembly_Trial_1.png‎|thumb|frame|center|No colonies were seen the next day.]]
-
 
+
'''Made more pSB1A3 stock'''
 +
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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|-
 +
|Trial || 260 || 280 || 260/280 || ng/μL
 +
|-
 +
|1 || 0.062|| 0.035 || 1.773|| 62.46
 +
|-
 +
|2 || 0.052|| 0.03 || 1.722|| 52.032
 +
|-
 +
|3 || 0.082|| 0.047|| 1.762|| 82.222
 +
|-
 +
|}
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Current revision

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December 6, 2012

Assemblies

  • 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

Dephosphorylation (11/23/12)

> Dephosphorylation (Roche)

  1. pSB1A3 - X/S = 2000 bp
Reagent Volume
DNA (clean digest) 11.0 (~200 ng)
10x phos. buffer 1.5
phosphatase 0.5
dH2O 2.0
  15 μL
  • Incubate at 37°C/ 10 min.
  • Heat inactivate at 75°C/ 2 min.
  • [final] = 200 ng/μL / 15 μL = ~13 ng/μL

Ligation

  • Use 20 ng of vector for each ligation = 1.5 μL pSB1A3
  • Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least 0.07 μL H2B
  • Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least 0.07 μL LOV
  • Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity
  H2B-LOV LOV-H2B Negative Control
H2B-LOV H2B insert DNA (1x mol vector) 1.0 μL 0 0
H2B-LOV LOV insert DNA (1x mol vector) 1.0 μL 0 0
LOV-H2B H2B insert DNA (1x mol vector) 0 μL 1.0 0
LOV-H2B LOV insert DNA (1x mol vector) 0 μL 1.0 0
Vector DNA (39 ng) 3.0 3.0 3.0
Gibson master mix 15.0 15.0 15.0
Total volume 20.0 20.0 18.0
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 50°C for 1 hour.

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

No colonies were seen the next day.
No colonies were seen the next day.

Made more pSB1A3 stock

Trial 260 280 260/280 ng/μL
1 0.062 0.035 1.773 62.46
2 0.052 0.03 1.722 52.032
3 0.082 0.047 1.762 82.222


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