Hi! I'm currently running 3C for an inter chromosomal interaction, and I have a few questions about the BAC control. Is it right if I purchase 2 BAC covering my regions of interest (on chromosome 6 and 10), and simply mix them together at equimolar amounts, followed by the usual digestion and ligation?
Do I have to clone the BAC myself, or is it okay to purchase commercially available BAC clones (I assume the BAC end pairs shown on UCSC genome browser are all commercially available?). I'm a beginner at cloning and will really appreciate any helpful input!
Hi Kay Leen,
It shouldn't be any difference between purchasing or cloning them by yourself, but obviously, they must contain the regions you are interested in. You then would cut the BACs with the restriction enzyme of your 3C libraries. Afterwards, the digested BACs will be mixed in equimolar amounts and re-ligated by T4 DNA ligase. The reason why you use the BACs is to evaluate the unspecific interactions between the two or more regions you are probing (unspecific ligation events that do not depend on the proximity of DNA strands). For this reason, you will analyse your primer pairs on the BAC control libraries and in the experimental libraries at equimolar concentration.
I hope that helps!
Hi Silvia Peluso thank you for your reply!
Instead of using BAC, do you think it will be possible for me to PCR amplify all the regions containing my primers and sequence them to check. Followed by mixing them in equimolar amounts and re-ligating them together for my control?
Hi Kay Leen ,
personally, I wouldn't do it. BAC libraries are used to determine differences in the efficiency of (PCR) oligos and efficiency of amplification. These libraries are generated by random ligation of restriction fragments (you need to digest the BAC with the same restriction enzyme) how would you overcome this step? The Digestion efficiency is a very important parameter too. I hope that helps!
Yes, Kay. You can use PCR products to generate de control library. In this case, you should design primers Foward and Reverse to amplify fragments containing the restriction site, purify them and mix in equimolar amounts. After that, follow with digestion and random ligation. To use it as a control library it is recommended you add to this purified template genomic DNA also digested with the same enzyme and ligated (separated tubes/reactions). This step will increase the complexity of the control library, making it more similar to 3C library.
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