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

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==11/21/12==
==11/21/12==
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'''Results from yesterday's ligation'''
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* Got 1:1 ratio of ligation: negative control for both
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* About 10 colonies total on every plate
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* Forgot to take into account X/S vector self-ligation
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* Will pick 4 colonies from each (H2B, LOV) to see if anything worked.
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* In the mean time, will retry cloning, trouble shoot procedure with help from Dr. Haynes
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 +
----
* Repeat PCR of H2B and LOV inserts
* Repeat PCR of H2B and LOV inserts

Revision as of 16:26, 21 November 2012

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11/21/12

Results from yesterday's ligation

  • Got 1:1 ratio of ligation: negative control for both
  • About 10 colonies total on every plate
  • Forgot to take into account X/S vector self-ligation
  • Will pick 4 colonies from each (H2B, LOV) to see if anything worked.
  • In the mean time, will retry cloning, trouble shoot procedure with help from Dr. Haynes

  • 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 ∞
  • Clean up PCR products using the Zymo Clean and Concentrator kit
  • Be sure to elute using 15 μL dH2O

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

Assemblies

  1. H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
  2. LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
  3. (negtaive control) pSB1A3 - X/S - 2000 bp
  • Digests:
    • In a 30 μL reaction, cut H2B with XbaI and SpeI
    • In a 30 μL reaction, cut LOV with XbaI and SpeI
Reagent H2B PCR LOV PCR
DNA 15.0 15.0
XbaI 1.0 1.0
SpeI 1.0 1.0
10x buffer 3.0 3.0
dH2O 10.0 10.0
Total vol. 30. 0 30.0


  • Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
  • Cut band out of gel (use UV box)


Results from gel recovery:

Sample 260 260/280 ng/μL
H2B - X/S ### ### ###
LOV - X/S ### ### ###


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

  1. H2B = 410
  2. LOV = 461


Dephosphorylation

> Dephosphorylation (Roche)

  1. pSB1A3 - X/S =
Reagent Volume
DNA (clean digest) up to 11.0 (~200 ng)
10x buffer d.p. 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 2x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL H2B = ### μL H2B
  • Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### ng/μL LOV = ### μL LOV
  H2B LOV Negative Control
Insert DNA (2x mol vector) ### μL ### ---
Vector DNA (50 ng) 1.5 1.5 1.5
2x Roche Rapid Ligation buffer 5.0 5.0 5.0
New England Biolabs T4 ligase 1.0 1.0 1.0
dH2O ### ### ###
TOTAL 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




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