I am new to Chip-chip, and have been following an Affymetrix protocol which uses random priming as the first step in amplifying the IP'd DNA. Other protocols use a blunt end/linker approach... any opinions as to which gives better amplification?
I had poor subsequent PCR amplification after 30 cycles, and after increasing to 60 I was just able to see the expected range of products on an agarose gel.
I am concerned that the random primer/adapter I am using has too much specific sequence and not enough 'randomness' (i.e. GTTTCCCAGTCACGGTC(N)9), resulting in poor linear amplification.

Hi,

Here are few hints that you may want to try in order to increase the yield of your genomic amplification.

1. the amount of initial Chip-ed material can be low and combining multiple experimental chip sample is a possibility.

2. More likely: the primer you are using is for a first round of random amplification (called round A). A second primer (called primer B in the original protocol) is then used. (same primer without (pdN)9. I usually never check the first round but rather at the second PCR round B for the presence of the smear.

I have been using the original protocol with success in the past (the original protocol can be found and downloaded at the following address: at http://cat.ucsf.edu/resources/index.html). Although we have slightly updated the protocol (check the supplementary protocol of this PloS publication ( PMID: 18076285; http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0050324).


note that the randomness is fine...

good luck