Team:UCLA/Notebook/Spider Silk Genetics/18 June 2015

iGEM UCLA




6/18/2015

BsaI digestion of VF/R Amplified MaSP

  • Digested 2x 1.5 ug of each DNA species in 50 uL with 4 uL BsaI. Follwed the following scheme.
AB (393) AB (393) BC (380) BC* (150) CA* (323) CA* (323)
10x Cutsmart 5 5 5 5 5 5
BsaI 4 4 4 4 4 4
DNA (1.5 ug) 3.82 3.82 3.95 10 4.66 4.66
ddH2O 37.18 37.18 37.05 31 36.34 36.34
Total 50 50 50 50 50 50
  • Incubated 50 C for 2hrs, heat kill at 65 C for 20 min.
  • Samples marked with asterisk were derived from samples from yesterday.

Results

  • cast 1.8 % gel to visualize. Used 2 uL 50 bp ladder.
Fig. 1 BsaI Digestion products of VF/R amplified MaSp. The band at 102 bp corresponds to the product of interest, and the 184 and 150 bp bands are residual.
  • The results of digestion reflect the poor PCR from yesterday. BC2 shows no DNA present at all, and both CA samples show decreased DNA. This problem is likely due to poor PCR purification.

Gel Extraction and Purification

  • Excised the 102 bp band.
  • Used Qiagen kit to recover gel DNA.
Concentration (ng/uL) A 260/280
AB 29.69 1.87
BC 28.81 2.55
CA 15.14 1.94

Solution Making

  • Preparation for BW buffer for ICA use.
  • Made 0.5 M EDTA pH 8 (ph with NaOH and HCl)
  • Made 1 M Tris-Cl pH 8 by adding 4N NaOH to to Tris-Cl pH 7.

Discussion with Sri

  • We should use e-gel to verify product after elution.
  • PCR amplify at different cycles using a dilution to also verify.
  • Using Magna-bind streptavidin beads is acceptable as a substitute for dyna-beads.