Matt:LabNotes/2015-3-14: Difference between revisions

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>Mzcai
(Created page with "==Design Primers for Sequencing Rolonies== *'''Purpose:''' Create sequencing libraries of rolonies to find which padlock probes of each gene contribute the most rolonies **Si...")
 
>Mzcai
 
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***P5+"GATCTACACGCCTGC"+RevComp(CL bases 1-19)
***P5+"GATCTACACGCCTGC"+RevComp(CL bases 1-19)
*SeqRolony_P7
*SeqRolony_P7
**CAAGCAGAAGACGGCATACGAGAT[Indx]TCCTCTGCCTCGGCGATATCCGACGGTAGTGT
**CAAGCAGAAGACGGCATACGAGATTCATGGTCCTCTGCCTCGGCGATATCCGACGGTAGTGT
***Tm = 59C
***Tm = 59C
***Indx =  
***Indx = TCATGG (Ind13)
***P7+"GAT"+Indx+"TCCTCTGCCTCGG+(CL bases 11-30)
***P7+"GAT"+Indx+"TCCTCTGCCTCGG+(CL bases 11-30)
*SeqRolony_RP1 (Read Primer 1)
*SeqRolony_RP1 (Read Primer 1)

Latest revision as of 23:18, 16 March 2015

Design Primers for Sequencing Rolonies[edit]

  • Purpose: Create sequencing libraries of rolonies to find which padlock probes of each gene contribute the most rolonies
    • Since all padlock probes of a gene have the same barcode it is impossible to tell which padlock probes are ligating and RCAing without sequencing H1/H2 gene

General Method[edit]

  1. Amplify in many (10-20) cycles of thermalcycling
    • Use primer annealing to common linker region to create many ssDNA of length >150bp which contains rolony barcode + H2 + H1
  2. Add Sequencing Adapters
    • Use 2-step PCR to add sequencing adapters
    • Must use common linker region for both but the 2 primers cannot be reverse complementary to avoid dimers
    • Instead allow 5' ends to overlap 5-8bp overlap

Primer Sequences[edit]

  • For Reference:
    • CA12k_Nov2014 Common Linker = CTTCAGCTTCCCGATATCCGACGGTAGTGT
    • P5 = AATGATACGGCGACCACCGAGATCTACACGCCTGC
    • P7 = CAAGCAGAAGACGGCATACGAGAT[Indx]CGGTCTGCCTTCCC
  • SeqRolony_CL_Amplify
    • TTCCCGATATCCGACGGTAGT
      • Tm = 63C
      • Shifted away from 5' end of Common Linker so that SeqRolony_P5 3' end won't anneal to the 5' end of product
  • SeqRolony_P5
    • AATGATACGGCGACCACCGAGATCTACACGCCTGCGGATATCGGGAAGCTGAAG
      • Tm = 60C
      • P5+"GATCTACACGCCTGC"+RevComp(CL bases 1-19)
  • SeqRolony_P7
    • CAAGCAGAAGACGGCATACGAGATTCATGGTCCTCTGCCTCGGCGATATCCGACGGTAGTGT
      • Tm = 59C
      • Indx = TCATGG (Ind13)
      • P7+"GAT"+Indx+"TCCTCTGCCTCGG+(CL bases 11-30)
  • SeqRolony_RP1 (Read Primer 1)
    • TACACGCCTGCGGATATCGGGAAGCTGAAG
      • Tm = 66C (IDT), 73C (Kun's)
  • SeqRolony_IRP (Index Read Primer)
    • ACACTACCGTCGGATATCGCCGAGGCAGAGGA
      • Tm = 68C (IDT), 76C (Kun's)