Team:UCLA/Notebook/Spider Silk Genetics/10 September 2015

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9/10/2015

Final Gel Image

• Redigest M2-9 and M1-9 using different sequence verified plasmids than yesterday.
• Digest using same conditions, enzymes
• Run on 1% TAE gel with 2 uL of NEB 1 kb ladder.

Fig. 1 Redone Final Gel image of ICA constructs. The incomplete digestion as well as the smear on M1-9 and M2-9 respectively are still present.

• The continued presence of the strange digestion patterns leads me to believe that this may be a problem that originates in the sequence of these plasmids. The possibility of two plasmid species is probably ruled out by clean sequencing reads.
• If we desire, we may repeat this image using XbaI and PstI to digest.

Protein Purification

• Grew 10 mL starter cultures for the 5 12-mers, which include the hybrid constructs.
• Inoculated M1-12(T7), M1/2[1:2]-12(T7), and M2-12(T7) into 1 L cultures.
• [2:1] and [1:1] did not grow very well, possibly due to age of plates.
• Streak M1/2[2:1]-12(T7) and M1/2[1:1]-12(T7) from glycerol stock for fresh colonies.