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

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(11/21/12)
Line 83: Line 83:
* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
* Cut band out of gel (use UV box)
* Cut band out of gel (use UV box)
 +
 +
Concentrations:
 +
...
Line 90: Line 93:
# H2B = 410
# H2B = 410
# LOV = 461
# LOV = 461
 +
Line 101: Line 105:
| <u>Reagent</u> || <u>Volume</u>
| <u>Reagent</u> || <u>Volume</u>
|-
|-
-
| DNA (clean digest) || up to 17 μL (500 ng)
+
| DNA (clean digest) || up to 11.0 (~200 ng)
|-
|-
-
| 10x buffer d.p. || 2.0
+
| 10x buffer d.p. || 1.5
|-
|-
-
| phosphatase || 1.0
+
| phosphatase || 0.5
|-
|-
-
| dH<sub>2</sub>O || ---
+
| dH<sub>2</sub>O || 2.0
|-
|-
-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
+
| &nbsp; || 15 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~13 ng/μL
|}
|}
 +
 +
* Heat inactivate ....
'''Ligation'''
'''Ligation'''
-
* Use 20 ng of vector for each ligation = '''1 μL pSB1A3'''
+
* Use 20 ng of vector for each ligation = '''1.5 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 H2B insert, relative to vector:  xμL  H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### 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'''
+
* 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 = '''0.8 μL LOV'''
{| class="wikitable" width=400px
{| class="wikitable" width=400px
| &nbsp; || H2B || LOV  || Negative Control
| &nbsp; || H2B || LOV  || Negative Control
|-
|-
-
| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || 0
+
| Insert DNA (2x mol vector) || ### μL || ### || ---
|-
|-
-
| Vector DNA (50 ng) || 1.0 || 1.0 || 1.0
+
| Vector DNA (50 ng) || 1.5 || 1.5 || 1.5
|-
|-
-
| 2x Roche Rapid Ligation buffer || 7.0 || 7.0 || 7.0
+
| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0
|-
|-
| New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0
| New England Biolabs T4 ligase || 1.0  || 1.0 || 1.0
|-
|-
-
| dH<sub>2</sub>O || 4.0 || 4.0 || 5.0
+
| dH<sub>2</sub>O || ### || ### || ###
|-
|-
-
| TOTAL || 14.0 || 14.0 || 14.0  
+
| TOTAL || 10.0 || 10.0 || 10.0  
|-  
|-  
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.

Revision as of 16:14, 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 products, 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

  1. H2B - X/S - 410 bp + 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 8.0 8.0
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)

Concentrations: ...


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 --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~13 ng/μL
  • Heat inactivate ....


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 = 1.28 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 = 0.8 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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