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

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

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

• 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:

1. H2B = 410
2. LOV = 461

Dephosphorylation

> Dephosphorylation (Roche)

1. pSB1A3 - X/S =

• 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

Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101