Hi,
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?
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Combining forward and reverse primer gives different size on gel
Started by jliao87, Jan 25 2011 02:10 PM
2 replies to this topic
#2
Maddie
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Posted 04 February 2011 - 10:10 AM
This is weird indeed. What was the mix prepared with? Buffer? water? How long and at what temperature was the mix stored?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.
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A. Einstein
#3
mdfenko
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Posted 04 February 2011 - 10:53 AM
are you sure that the f/r mix was using the same f and r as the individual primers?
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genius does what it must
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