Sunday 05/05/13

STORM Analysis

• Fixed bug in RunDotFinder that was causing DaoSTORM analysis on to correctly overwrite/rerun analysis (DaoSTORM method check had method in quotes, causing failure).

Project 2

• Data analysis notes

OligoPaints

• Running 2.5 mL of PCR using PPTC primer at 60C for B,Y,K and G libraries.
• Does not look to good on gel:
  
• Blue failed again! This worked so well with inner primers yesterday:
  
• Set up new PCR gradient 50:70C (12 steps) using PPTC and sublibrary-specific NP primers
• Set up second gradient, added 1.5 uL PCR1 product (amplified with inside primers) and adapter primers + NP primers (+ PPTC from original dilution). This one 55:72C gradient (12 steps), 15 uL reactions.

Qubit

• All measurements in ug/mL

Nanodrop

• in ug/mL

Column clean up 2.5 mL scale

• 15 mL (2.5 + 12.5 binding buffer) is just about full volume capacity for the CC-100 columns. (this is true according to spec sheet).
• Spinning at 3,000g with 15 mL on top breaks columns (can be snapped back together)
• Spinning at 300g gets the 15mL through, then need a short spin at 14,000g in mini-centrifuge to elute remaining binding buffer.
• 2x 2mL wash on manifold
• spin dry at 13,000 rpm
• elute with 150 uL 50C ddH2O.

Orders

• # Order more primers
• # Order more probe synthesis reagents + troubleshooting diagnostics.