Combining forward and reverse primer gives different size on gel
Posted 25 January 2011 - 02:10 PM
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?
Posted 04 February 2011 - 10:10 AM
Posted 04 February 2011 - 10:53 AM
genius does what it must
i do what i get paid to do