Team:UCLA/Notebook/Spider Silk Genetics/5 June 2015

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6/5/2015

ICA Oligo Characterization

• The results from 6/3/2015 showed that C-cap could bind to MaSp2 CA.
• We want to find out which sticky end the c-cap binds to.
  • If the C-sticky end is bound, then there should be no problem.
  • If the A-sticky end is bound, it could possibly interfere with ICA by competing with binding to MaSp2 AB.
• We also decided to test the initiator and capping oligos again, but at a higher concentration.
• We set up the following ligation reactions.
  • 2 reactions each of BC+CA and CA+AB were set up using 50 ng of each fragment.
  • AB+Init and CA+Term were set up using 1 uL of 5 uM oligo in each.
  • Used T7 polymerase for all 6 reactions. Incubated for 10 minutes at 25 C.
  • After incubation, 1 uL of 5 uM working C-Cap was added to one BC+CA reaction and one CA+AB reaction each, and incubated for another 10 minutes at 25 C.

Results

• Cast 2.5 % TAE gel for visualization, used 2 uL of 50 bp ladder.

Fig. 1 The BC+CA reactions with or without C-cap show no double band character at the 200 bp mark. The CA+AB+C-cap reactions exhibits a double band similar to the CA+C-cap reaction from 6/3/2015. AB+init exhibits a band at approximately 160 bp. CA+term exhibits a band at approximately 140 bp.

• From these results, we can conclude that C-cap binds to the C-sticky end, and not the A-sticky end. We will be able to proceed with ICA as planned.
• The initiator and terminator work as intended. The expected band sizes are 161 bp and 141 bp respectively.
• All bands smaller than 100 bp correspond to unligated oligos.
• In addition, there is a faint band at about 450 bp in the CA+AB reactions. We currently have no explanation for this, but the band size is similar to undigested MaSp2 that was amplified with VF/R primers.