Combining forward and reverse primer gives different size on gel - (Jan/25/2011 )
I recently ran a RT-PCR protocol. RNA was isolated using Qiagen RNeasy mini kit, reverse transcription and primer annealing done via Superscript III.
The primer I used had been around for awhile but the stock was stored correctly. Someone (from before I arrived at the lab) had prepared a vial of the same primer but with the forward and reverse primers combined.
I ran it separately; one lane was F and R primers from stock, the other lane was the pre-mixed f/r primers.
I viewed the gel and found that the lane in which I added the primers individually was in the correct approximate size (196bp). However, the mixed stock resulted in an intense band at ~400bp and none at ~200bp.
What causes (or can cause) primers mixed together to do this?
This is weird indeed. What was the mix prepared with? Buffer? water? How long and at what temperature was the mix stored?
are you sure that the f/r mix was using the same f and r as the individual primers?