Matt:LabNotes/2015-3-14
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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]
- 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
- 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
- TTCCCGATATCCGACGGTAGT
- SeqRolony_P5
- AATGATACGGCGACCACCGAGATCTACACGCCTGCGGATATCGGGAAGCTGAAG
- Tm = 60C
- P5+"GATCTACACGCCTGC"+RevComp(CL bases 1-19)
- AATGATACGGCGACCACCGAGATCTACACGCCTGCGGATATCGGGAAGCTGAAG
- SeqRolony_P7
- CAAGCAGAAGACGGCATACGAGATTCATGGTCCTCTGCCTCGGCGATATCCGACGGTAGTGT
- Tm = 59C
- Indx = TCATGG (Ind13)
- P7+"GAT"+Indx+"TCCTCTGCCTCGG+(CL bases 11-30)
- CAAGCAGAAGACGGCATACGAGATTCATGGTCCTCTGCCTCGGCGATATCCGACGGTAGTGT
- SeqRolony_RP1 (Read Primer 1)
- TACACGCCTGCGGATATCGGGAAGCTGAAG
- Tm = 66C (IDT), 73C (Kun's)
- TACACGCCTGCGGATATCGGGAAGCTGAAG
- SeqRolony_IRP (Index Read Primer)
- ACACTACCGTCGGATATCGCCGAGGCAGAGGA
- Tm = 68C (IDT), 76C (Kun's)
- ACACTACCGTCGGATATCGCCGAGGCAGAGGA