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

(Difference between revisions)
 Revision as of 17:20, 21 November 2012 (view source) (→11/21/12)← Previous diff Revision as of 17:23, 21 November 2012 (view source) (→11/21/12)Next diff → Line 46: Line 46: '''Assemblies''' '''Assemblies''' # H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp # H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp + # LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp + # (negtaive control) pSB1A3 - X/S - 2000 bp * Digests: * Digests: Line 119: Line 121: '''Ligation''' '''Ligation''' * Use 20 ng of vector for each ligation = '''1.5 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 = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / ### 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 = '''### 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''' + * 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''' {| class="wikitable" width=400px {| class="wikitable" width=400px

## Revision as of 17:23, 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 ∞
• 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 ### ### ###

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