I’m using some of the Moerman Lab / Geneservice fosmids and would like to know what your experience has been regarding yield. I grew up and induced 17 fosmids using the 1 ml culture directions. I used a standard Promega miniprep kit, eluting in 75 ul. Concentrations for most were 4-6 ng / ul as read on a Nanodrop spec. Only four were higher than 10 ng / ul and one was only 1.9 ng / ul (almost nothing). Is this yield consistent with what others have seen? Do you normally have to do the 5 ml induction culture to get a decent amount?
I think that the concentrations you obtained are within the normal range. The fosmids are single copy, so you just won’t get much from small minipreps. You can obviously grow up larger preps, or you can transform into EPI300 cells from epicentre and use their autoinduction solution to induce multiple copies of the fosmid. I’ve also used Macherey-Nagel Nucleobond PC 20 minipreps from 10 mL of culture and obtained enough fosmid to work with and sequence. Promega wizard midipreps (100mL culture) work as well.
The fosmids are low copy number, and also many of the mini-prep kits do not work well with large plasmids. My lab has found that the Epicentre FosmidMAX kit works best (in comparison with the Qiagen spin mini-prep and Qiagen maxi-prep kit) for small scale and larger scale preps. The EPI300 bacteria are really helpful for increasing the yield with the larger scale prep.
I was not aware you had to retransform the fosmids into another strain to do induction. The directions on the Geneservice web page make it seem like you can induce them in the cells in which the library is provided. I used the regular induction solution from Epicentre and not the autoinduction protocol because that is what Geneservice recommends. Do you get a higher yield with autoinduction?
Thanks for your replies and advice about the other kinds of prep kits.
I found out that the fosmid library clones are already in EPI300 cells and can be induced directly.
I am making preps with the FosmidMax kit as you suggested. Do you do the standard protocol or the one that gets rid of RNA earlier on. I’m going to use the fosmid DNA to make transgenic lines and want to be sure that the residual RiboShredder solution won’t interfere with this process. Also, I assume that the reason Epicentre suggests quantitation by fluorimetry is to negate the effect of residual rNTPs. Let me know if anyone has successfully done quantitation of these preps with a NanoDrop (which we have).
you need to induce the high number of copy with a solution of L arabinose 10 % that you dilute 1000X. I am doing usually the induction overnight from a one colonie culture (do not leave more than 16 hours). After I am using a Qiagen kit but I think whatever kit you want should be find
We use the protocol that removes the RNA early on for the very reason you suggest. Afterwards, we are able to quantify the DNA concentration either via a regular spectrophotometer or a Nanodrop with good results. For injection, probably the small scale prep is fine, but for bombardment we use a 50 ml prep which gives enough DNA for multiple bombardments.