Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/07

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December 7, 2012

  • Results from 12/6/12 ligation was unsuccessful
  • Will try regular pSB1A3 cut with XbaI and SpeI instead of dephosphorylated

Digestion

  • In a 30 μL reaction, cut a pSB1A2 plasmid (from Rene) XbaI and SpeI, then ran on 1% gel and used Zymoclean Gel DNA Recovery Kit
Reagent pSB1A2
DNA 15.0
XbaI 1.0
SpeI 1.0
10x buffer 3.0
dH2O 10.0
Total vol. 30.0

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

Results of the enzyme digest is shown, with both pSB1A3 bands on the right. The gel was loaded with 30 μL samples. The bands that were cut are outlined in green.

  • Results from gel recovery
Sample 260 280 260/280 ng/μL
pSB1A3 0.007 0.004 1.711 6.916

Ligation

  • Use 20 ng of vector for each ligation = 3 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, the inserts were rounded to 1 μL for simplicity
  H2B-LOV Negative Control
H2B-LOV H2B insert DNA (1x mol vector) 1.0 μL 0
H2B-LOV LOV insert DNA (1x mol vector) 1.0 μL 0
Vector DNA (39 ng) 3.0 3.0
Gibson master mix 15.0 15.0
Total volume 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

There were some pseudo colonies seen the next day but these had a white film so they probably did not contain the ligation.
There were some pseudo colonies seen the next day but these had a white film so they probably did not contain the ligation.



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