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

(Difference between revisions)
Jump to: navigation, search
 Revision as of 16:07, 21 November 2012 (view source) (→11/21/12)← Previous diff Revision as of 16:14, 21 November 2012 (view source) (→11/21/12)Next diff → 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: | Reagent || Volume | Reagent || Volume |- |- - | 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 |- |- - | dH2O || --- + | dH2O || 2.0 |- |- - |   || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL + |   || 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 |   || H2B || LOV  || Negative Control |   || 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 |- |- - | dH2O || 4.0 || 4.0 || 5.0 + | dH2O || ### || ### || ### |- |- - | 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.
Incubate at room temperature for 10 minutes. | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.

## Revision as of 16:14, 21 November 2012

Project name Main project page
Previous entry      Next entry

## 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 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: ...

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