Hi Folks
Iam using Tru seq small RNA prepatation kit for library construction and observe very faint band of small RNA library .
MY ISSUE:
I pooled my 8 plant small RNA libraries and run them on a 6% TBE gel and I observed the desired micro RNA band of 147 and 157 nt are not clear and sharp and they are very faint.
1. I used 2.5ug of small RNA as input material,will using more than 1ug of small RNA as starting material have any problem?.My small RNA sample used for this protocol was intact and not degraded.
2. I tried to run PCR with maximum cycles recommended (15 cycles)
3. Will pooling only 5 ul of each sample with 8 different indexes and loading them on gel result in this faint band/low amount of library?
Please advise me on this
thankyou
Jay
Iam using Tru seq small RNA prepatation kit for library construction and observe very faint band of small RNA library .
MY ISSUE:
I pooled my 8 plant small RNA libraries and run them on a 6% TBE gel and I observed the desired micro RNA band of 147 and 157 nt are not clear and sharp and they are very faint.
1. I used 2.5ug of small RNA as input material,will using more than 1ug of small RNA as starting material have any problem?.My small RNA sample used for this protocol was intact and not degraded.
2. I tried to run PCR with maximum cycles recommended (15 cycles)
3. Will pooling only 5 ul of each sample with 8 different indexes and loading them on gel result in this faint band/low amount of library?
Please advise me on this
thankyou
Jay
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