Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/23

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November 23, 2012

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 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
  • 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


  H2B LOV Negative Control H2B LOV Negative Control
Insert DNA (2x mol vector) 1.0 μL 1.0 --- 1.0 1.0 ---
Vector DNA (50 ng) 1.5 1.5 1.5 1.5 1.5 1.5
2x Roche Rapid Ligation buffer 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase 1.0 NEB 1.0 NEB 1.0 NEB 1.0 Roche 1.0 Roche 1.0 Roche
dH2O 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
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


Digestion

  • Check ligations from 11/20/12 to see if any colonies contained the insert
  • Used 4 colonies from H2B, 4 colonies from LOV plates
  • Each colony liquid culture followed the digestion table accordingly
Reagent H2B LOV
DNA 5.0 5.0
EcoRI 1.0 0
PstI 0 1.0
10x buffer 1.5 1.5
dH2O 7.5 7.5
Total vol. 15.0 15.0
  • Run entire 15 μL on 1% gel
  • Expect 2 bands for H2B and 3 bands for LOV

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

Results of the enzyme digests are shown, with four H2B bands on the left, and three LOV bands on the right. The gel was loaded with 30 μL samples. The bands are at 2000 bp, confirming the vector self ligated and there is no insert.



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