Matt:LabNotes/2014-1-30

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Revision as of 02:08, 1 February 2014 by >Mzcai (Created page with "===Block Shifted === *Block Shifted FISSEQ_2ndRCAprimerDye with RT primer oligonucleotide to see if they are hybridizing to the same locations on primary rolonies ====Procedu...")
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Block Shifted

  • Block Shifted FISSEQ_2ndRCAprimerDye with RT primer oligonucleotide to see if they are hybridizing to the same locations on primary rolonies

Procedure

  1. Stripped with 80% formamide for 10min at 50C
  2. Hybridize RT primer 1uM (2ul 100uM Dye + 60ul 100% formamide + 20ul 20X SSC + 118ul H2O)
  3. Wash with 2X SSC twice
  4. Hybridize Shifted FISSEQ 2uM (2ul 200uM Dye + 60ul 100% formamide + 20ul 20X SSC + 118ul H2O)
  5. Wash with 2X SSC twice
  • In the future, the blocking oligonucleotide should be atleast one order of magnitude greater in concentration

Results

24hrs Blocked
289 107
116 68
359 366
21370 3213