Team:UCLA/Notebook/Spider Silk Genetics/21 July 2015

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7/21/2015

Colony Results

• Results of yesterday's transformations.
• Glycerol plates all have colonies.
• M2-9(T7) has very small colonies.
• M2-12(1C3) has no colonies.
  • Will redo PCR, ligation, transformation.

PCA of MaSp1 AB, BC, CA

• Scaled up the protocol from yesterday for 50 uL reactions for MaSp1 AB, BC, CA each.
• We used 64 C as the annealing temperature.

PCR of 12-mer

• Starting from PCR of 12-mer due to transformation failure.
• We could re-transform with the leftover ligation, but we are not certain if it is good.
• Repeated protocol from 7/16/2015.
  • Scaled to 1x 50 uL reaction.

Results of MaSp1 PCA, 12-mer PCR

• Cast 1.5% TAE gel to visualize, used 2 uL of 100 bp ladder.
• Ran 5 uL of the MaSp1 PCR to verify presence of desired product.

Fig. 1 Assembly of MaSp1 AB, BC, CA. PCR amplification of M2-12-mer. The expected product for MaSp1 is 170 bp. The expected product for M2-12-mer is 1324 bp. There is some off-target amplification present in the 12-mer sample.

• Results indicate that the PCA of MaSp1 was successful.
• Results indicate that PCR of the M2-12-mer was also successful.
• Excised the bands corresponding to the M2-12-mer.
• Gel-purified: 20.38 ng/uL

Digestion of MaSp1 AB, BC, CA and M2-12-mer for cloning

• Digest with EcoRI, PstI for cloning into pSB1C3.
• Also digested some linearized backbone.

	Backbone (25 ng/uL)	M1-AB (179.98 ng/uL)	M1-BC (262.32 ng/uL)	M1-CA (181.57 ng/uuL)
EcoRI	1 uL	1 uL	1 uL	1 uL
PstI	1 uL	1 uL	1 uL	1 uL
10x 2.1 NEBuffer	5 uL	5 uL	5 uL	5 uL
DNA (~1 ug)	24 uL	5.6 uL	3.8 uL	5.5 uL
ddH2O	19 uL	37.4 uL	39.2 uL	37.5 uL
Total	50 uL	50 uL	50 uL	50 uL

• Digest 37 C 1.5 hrs, heat kill 65 C 20 min.
• Post-digestion: PCR clean-up using Zymo Clean and Concentrator.

Ligation

• Ligate M1 AB, BC, CA and M2-12(E,P digest) into pSB1C3(E,P digest).

	M-12 (20.38 ng/uL)	M1-AB (36.82 ng/uL)	M1-BC (27.82 ng/uL)	M1-CA (37.53 ng/uL)
10x T4 Ligase Buffer	2 uL	2 uL	2 uL	2 uL
pSB1C3 (50 ng)	1.4 uL	1.4 uL	1.4 uL	1.4 uL
Insert	4.4 uL	1.4 uL	1.8 uL	1.3 uL
ddH20	11.2 uL	14.2 uL	13.8 uL	14.3 uL
Total	20 uuL	20 uL	29 uL	20 uL

• Ligate 25 C for 25 min, heat kill 65 C 10 min.

Transformation

• Transform M2-12(1C3), M1-AB, BC, CA into electrocompetent DH5(alpha) cells.
• Arc times:
  • M2-12(1C3): 5.4
  • M1-AB: 5.6
  • M1-BC: 5.6
  • M1-CA: 5.6
• Plated all the cells.