Hosuk:LabNotes/2013-12-30
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RT primer annealing specific position in mRNA
Primer Design
- Target gene : RAB7A
- RAB7A mRNA target sequence: CAGAACTTGGACCTTCTCGCTTCTGTCCTCCGTTTAGTCTCCTC
- This is base 125-177
- Choose primers that are 10bp long while minimizing GC content and ~200bp, ~300bp, ~500bp, and ~1000bp from TSS
- mRNA target of RAB7A 220b distance from TSS : CGCGTTTGAA
- mRNA target of RAB7A 300b distance from TSS : ACTCATGAAC
- mRNA target of RAB7A 510b distance from TSS : CAACACATTC
- mRNA target of RAB7A 1000b distance from TSS: TTACACCCCA
- RT_RAB7A_220b: [/5Phos/TCTCGGGAACGCTGAAGA] + TTCAAACGCG (--> D220)
- RT_RAB7A_300b: [/5Phos/TCTCGGGAACGCTGAAGA] + GTTCATGAGT (--> D300)
- RT_RAB7A_510b: [/5Phos/TCTCGGGAACGCTGAAGA] + GAATGTGTTG (--> D510)
- RT_RAB7A_1000b: [/5Phos/TCTCGGGAACGCTGAAGA] + TGGGGTGTAA(--> D1000)
Rolony Fab.
- Use 96 well plate
- Control : Random Hexamer