Hi all,
I am new to the Illumina system. When I did the PCR and ran the gel using normal sets of primers. Everything is nice and clean. However, when I used the primers with Illumina adapters. I got non-specific bands and smear. Can someone help me please?
PCR conditions are the same for both with/without the adapters.
16S primers: 515'F/926r
GTGBCAGCMGCCGCGGTAA
CCGYCAATTYMTTTRAGTTT
16S primers with adapters:
5'[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTGBCAGCMGCCGCGGTAA-3'
5β[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]CCGYCAATTYMTTTRAGTTT-3'
16S PCR condition:
95 C - 5 min
35 cycles of 95 C - 30 sec/ 55.5 C - 30 sec / 72 C - 30 sec
72 C - 10 min
18S primers: Euk1391f/EukBr
GTACACACCGCCCGTC
TGATCCTTCTGCAGGTTCACCTAC
with adapters:
5β[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTACACACCGCCCGTC-3'
5β[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]TGATCCTTCTGCAGGTTCACCTAC-3'
18S PCR condition:
94C - 3 min
35 cycles of 94C - 45 sec/ 57C - 60 sec/ 72C - 90 sec
72C - 10 min
These are all environmental DNA which the original extracted DNA concentration are very low.
Thank you!
Kitty
I am new to the Illumina system. When I did the PCR and ran the gel using normal sets of primers. Everything is nice and clean. However, when I used the primers with Illumina adapters. I got non-specific bands and smear. Can someone help me please?
PCR conditions are the same for both with/without the adapters.
16S primers: 515'F/926r
GTGBCAGCMGCCGCGGTAA
CCGYCAATTYMTTTRAGTTT
16S primers with adapters:
5'[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTGBCAGCMGCCGCGGTAA-3'
5β[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]CCGYCAATTYMTTTRAGTTT-3'
16S PCR condition:
95 C - 5 min
35 cycles of 95 C - 30 sec/ 55.5 C - 30 sec / 72 C - 30 sec
72 C - 10 min
18S primers: Euk1391f/EukBr
GTACACACCGCCCGTC
TGATCCTTCTGCAGGTTCACCTAC
with adapters:
5β[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTACACACCGCCCGTC-3'
5β[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]TGATCCTTCTGCAGGTTCACCTAC-3'
18S PCR condition:
94C - 3 min
35 cycles of 94C - 45 sec/ 57C - 60 sec/ 72C - 90 sec
72C - 10 min
These are all environmental DNA which the original extracted DNA concentration are very low.
Thank you!
Kitty
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