Wednesday 09/11/13

9:30a – 11:30p

Review

• submit review
• submit copyright agreement

BIRS

• draft email of final reminders
• contact Vistas Dining hall about meal costs and reserving tables
• Print driving directions to and from Airport/Banff, Lakes, and hotel.
• Print hotel receipt

Probe making

• Gel, check PCRs
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• bleach all pipettes
• New test PCR:
  1. Abd-B T7 + common + lib 1:20
  2. Abd-A T7 + common + lib 1:20
  3. no reverse + common + lib 1:20
  4. E6 positive control + common + lib 1:20
     

STORM analysis

• Run splitQVdax on G4 data

Planning: QC for library: deep sequencing

option 1

Amplify sublibraries
* Buy NEB DNA kit, end repair
* blunt end ligation of illumina primers

option 2

• PCR primer
• gel-extraction
• Qubit concentration (nanodrop?) [optional]
• BioAnalyzer at Bauer Core. [optional]
• qPCR, dilute sample a few times. By Illumina qPCR kit from kappa.
• Mx3000p (less used) qPCR machine at Bauer core. On every sublibrary with my index.
• use this concentration to dilute to 10 nM single strand. Combine and submit.
• (triple measure of concentration).
• low complexity sequence problem for reader.
• which primer gets read from.

Adapter sequences

• Index Primer 5′-3′: AGT TCA GAC GTG TGC TCT TCC GAT C
• Universal Primer 5′-3′: CC CTA CAC GAC GCT CTT CCG ATC T
• one of these gets 5-random-Ns + common primer added to the end. (the one which contains the sequencing primer)
• The other gets T7 sequence added to the end.
• I have written to NEB to find out which sequence contains the sequencing primer.

Recent probe making

• Yellow probes: E6 E7 E10 F6