Team:UCLA/Notebook/Spider Silk Genetics/20 August 2015

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8/20/2015

Size Verification for M1-12(1C3)

• Digested 1 ug of each of the sequence verified plasmid from yesterday with EcoRI and PstI in a 50 uL reaction.
• Used NEBuffer 2.1
• Digest at 37 C for 1.5 hours, heat kill at 65 C for 20 min.
• Run 15 uL of digest on 1% TAE gel, with 2 uL of NEB 1 kb ladder.

Fig. 1 Size Analysis of Putative M1-12(1C3). The expected size for a digested 12-mer is 1324 bp. The expected size for a digested 9-mer is 1018 bp. The expected size for digested pSB1C3 is 2029 bp. The slight size difference ins sequence sample 4 is due to the inclusion of the T7 promoter-RBS sequence from BBa_K525998.

• Results indicate that what we believed to be M1-12(1C3) is in reality M1-9(1C3). This results is most likely due to contamination during the transformation process, possibly from the glass beads used to spread bacteria.

Maxi-Prep for M2-AB, BC, CA

• Used Qiagen Maxi kit to prepare M2-AB, BC, CA.
  • Eluted in 200 uL EB.

	Concentration	A 260/280
M2-AB	547.33	1.87
M2-BC	587.63	1.88
M2-CA	525.92	1.87

BsaI Digestion for M2 Monomers

• Digested 5 ug of each monomer plasmid in 50 uL using 4 uL of BsaI. Digested 2x 50 uL reactions for each.
• Digest at 50 C for 2 hours, heat kill at 65 C for 20 min.
• Ran on 1.5% TAE gel, with 2 uL of NEB 50 bp ladder.

Fig. 2 BsaI Digestion of M2 monomers. The expected size of digestion product is 102 bp.

• Excised the 102 bp band for gel extraction.