Team:UCLA/Notebook/Spider Silk Genetics/26 June 2015

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6/26/2015

• A re-reading of Briggs et al. indicates that the amount of initiator used was very small. (5 uL of 10 nM initiator)
• Our previous experiments used much more initiator (1 uL of 5 uM initiator)
• Excess initiator may be interfering with PCR, or binding of pieces, so we decided to repeat ICA testing using reduced initiator.
• We decided to test making IABCT with and without caps, and IT using less initiator this time compared to 6/19/205.

ICA with 5 uL of 10 nM initiator

1. Used 3x 10 uL of MagnaBind Beads.
   • washed 2x using 2x BW buffer.
2. Incubated in a 10 uL reaction containing:
   • 5 uL of 2x BW buffer.
   • 1 uL of 50 nM initiator.
   • 4 uL of ddH2O.
3. Incubate at RT (~23 C) for 45 min with rotation.
4. Subsequent steps followed the same protocol as 6/19/2015 with the following modifications:
   • For one ICA reaction, no caps were used, and were instead substituted with water.
   • A-caps, however, were used for both IABCT reactions in order to reduce formation of IT construct.
   • Washes were performed using 100 uL of solution, rather than 15 uL.
   • Final elution was performed at 95 C for 5 min, rather than 3 min.

PCR Amplification of ICA

• Set up 25 uL reactions for each ICA reaction. Used 1 uL of eluate as template.
• Same protocol as yesterday.
  • Modified to anneal for 25 cycles, instead of 20.

Results

• Cast 1.5% TAE gel, used 2 uL of 100 bp ladder.

Fig. 1 Amplification of ICA with reduced initiator. The expected size for IABCT amplification is 406 bp. The expected size for IT amplification is 100 bp. There are faint bands present at 100 bp for all three samples. There is a smear starting at 400 bp for all samples.

• Results are inconclusive. The beads appear to bind to the biotinylated IT, but not to IABCT.
• The smear at 400 bp is present in all samples, and is not likely to be IABCT.