random hexamers for cDNA synthesis
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Aug 15 05:01:26 EST 2000
In article <Pine.PMDF.3.96.1000814174237.551644504A-
100000 at sask.usask.ca>, brownlie at sask.usask.ca writes
>I understand that random hexamers are often used to prime first strand
>synthesis to produce cDNA and that this method often results in longer
>products and more representative libraries than with oligo dT.
>From our experimental data random hexamers do not give us the longest
products. Specific primers or oligodT work best. Random hexamers may
give more representative libraries in that they will primer mRNA where
the polyA tail is insufficient length for oligodT to anneal to. Random
hexamers by their non-specificity will of cause prime rRNA.
> However, I
>am confused what the theory is behind this as will random hexamers not
>result in many truncated products and what stitches these together before
>second strand synthesis.
Nothing stitches them together. You are relying on the reverse
transcriptase to transcribe as long a length as possible. You
automatically generate overlapping, truncated products and there is
nothing you can really do about it.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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