Dear Cash,

Said that I think is better a 4C approach for identifying possible intra-domain contacts within the same TAD (topological associated domain), I don't understand completely the question.

Do you need to know how to design primers in general, or are you interested in knowing how to pick up enhancers locations to use as viewpoints for your assay?

hi valerio,

i am interested in knowing strategy to designe primers for this 3c experiment of course when u know the location of enhancer for a particular gene.

more over to this i am interested  in knowing  how predict that a particular genomic location is playing role as enhancer for a gene x. because then only you can plan your 3c experiment keeping in mind that that these are tissue specific.

yes, i am agree with your viewpoint regarding 4C.

Dear Cash,

There are many ways to design a primer. we a specific species (plant or animal) to help you more in detail. If have accession number, you can design a primer using the following web tools (free)

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://simgene.com/Primer3

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/

https://tools.thermofisher.com/content.cfm?pageid=9716&icid=fr-oligo-6?CID=fl-oligoperfect

https://www.genscript.com/ssl-bin/app/primer

Good luck

dear Tewodros Endalew,

these are conman primer designing tools . but i am asking primer designing strategies for a particular experiment that is- 3C (chromatin conformation capture)

Dear Cash,

Check these documents if it helps to answer your question and the link below:

http://www.abcam.com/protocols/getting-started-with-chromatin-conformation-capture-3c

Good luck

Hi Cash

you can use primer design tool like Primer Express Software from ABI to design a series of primers with similar melting temperatures and PCR efficiencies that bind next to the restriction sites analyzed by 3C. For locus-wide analysis of the association of a particular site, a common primer (anchor primer) adjacent to this site can be used in combination with specific primers for each of the sites tested for an association. Since two sites are scored as being associated when their association frequency is higher than those of intervening sites, it is important to design primers also for testing the intervening region. It is also important to test genome-wide associations in a reciprocal manner by setting the anchor primer in one of the elements previously tested for association. This is a control to ensure the validity of the detected associations

ok shishir , let us just discuss about it , please manage timings for this.

to Salwa Wahsh - please describe in detail , and how to select a particular restriction enzyme in this case ?

In my lab we mostly use DpnII and Csp6I as first and second cutter. The difference is the level of detail you want to resolve. DpnII is a 4 bp cutter (^GATC), and has a frequency on the genome of a cut every 200-300 bp. If you use a 6bp cutter like HindIII, then you restriction fragments will be longer, thus the resolution will be lower. Usually you will use a 6bp cutter in case you want to identify mostly TAD structures, while with a 4bp cutter you can pinpoint the local interactions of your specific viewpoint.

thank you Valerio Bianchi, i am thankful for this reply, if you have any paper to describe application of 4bp or 6bp cutter in much details please let me know

Hi, Valerio Bianchi

Nice to see your detail response to this discussion. Do you guys also performed 4C experiments? If so I will be grateful to have a detail protocol. Thanks

Late to the party but I'm struggling with this at the moment.

I'm trying to design primers for 4C with primer3, but I'm not sure it's a suitable tool; it seems to require some optimisation at least. I got some primers, but the problem is that they're suite for traditional PCR (i.e. they'd amplify the bait locus, which is non-informative), whereas for 4C I need to primers to amplify away from the bait locus into the interacting fragment.

There's an online tool for that (https://mnlab.uchicago.edu/4Cpd/) but it doesn't have my first RE (ApoI) in the list.

Any tips are welcome!

Cheers!

Bernardo Moreira Soares Oliveira for my last 4C (35 viewpoints) I used primer3 in combination with AmplifX software. Basically I just choose the forward and the reverse primers separately in primer 3 since you want inverse PCR so not facing primers (if you get what I mean). I used the qPCR settings of primer 3, trying to use very close Tm between primers of a same Viewpoint.

My advice is to design several primers for adjacents fragments you want to look, because there will always be not working primers (I had apprx 20% ofall my disigned primers leading to non-linear amplification). You should carefully check for amplification efficiency of your primers for inverse PCR.

Another tip, don't use ApoI except it's the one cutter you need for your viewpoints. I never got good results in 3C at least. It doesn't cut really well..

Hope it helps !