I am new grad student having trouble amplifying a library (5E6 clones) for a 2-hybrid experiment in yeast. I am currently using Qiagen's Midi kit for isolation of the library plasmids (once amplified in E. coli) and am still getting 5 ng/ul of DNA and messy 260/280 ratios. How can I increase my yield/purity (using this kit?)? Also, what other protocols out there exist for 2-hybrid library amplifications (in E. coli)?
Thanks!!!

I'm not familiar with the specifics of a 2 hybrid library, but I've amplified a lot e. coli libraries and purified using the Q. Midi Kit. One of the biggest problems I had was overloading the columns. Try splitting the starting volume of cells in 2 or 3 seperate volumes and purifying each seperately and mixing the library preps after purification.

You can always precipitate the library DNA later.

vasussci said:

I'm not familiar with the specifics of a 2 hybrid library, but I've amplified a lot e. coli libraries and purified using the Q. Midi Kit. One of the biggest problems I had was overloading the columns. Try splitting the starting volume of cells in 2 or 3 seperate volumes and purifying each seperately and mixing the library preps after purification. You can always precipitate the library DNA later.

I did notice that the columns got very full with lysate (which remained somewhat viscous and brown still). It is possible that with too many cells (or too much DNA) the system is simply overloaded and I am not getting anything through? Thanks.

I did notice that the columns got very full with lysate (which remained somewhat viscous and brown still).

Your problems can probably be worked out by reading pages 63-73 of the booklet that comes in any qiagen plasmid prep kit.

The Qiagen Kits should be perfect for what you are doing. However it is a common mistake to overgrow your E.Coli cultures. It also sounds like you are not lysing all your sample or neutralizing it properly. This leads to 2 problems.

1) Overgrowing or using too much culture saturates the capacity of the alkaine lysis step. If you continue to grow the same amount of bacteria, use more P1, P2 and P3 to lyse the cells (increase in equal proportions). Qiagen now sell a product called "LyseBlue" to use with their plasmid prep kits (http://www1.qiagen.com/Plasmid/LyseBlueInfo.aspx). This tells you if the lysis step has gone to completion or not. Somewhat of an unnessessary extra step in my book, but if you are new to plasmid preps then why not try it? It cannot hurt if you are having problems.

Also, if there is any residual SDS left over after neutralization, it will inhibit binding of the plamid DNA to the resin in the columns. Add a little more P3 than the manual says if you think this might be a problem.

2) Assuming you have lysed all or most of your sample, you can overload the resin in the column if you started with too many bugs. Also make sure none of the crud from the lysis step gets into your column. Eluting DNA from clogged columns is not easy.

Check the copy number of your plasmid in you host E. Coli strain. If it is a high copy number plasmid, purify less culture, or just grow smaller cultures. You can always check your cell density my taking an OD600 on a spec.

Check your host bacterial strain - JM101, JM110, HB101 etc have high endonuclease activity and this can lead to poor DNA quality at the end - Use one of the Endo free kits for these strains.

Changing your host strain to DH5alpha or XL1 Blue may help.

Oh, and if you are having these kind of problems perform those extra steps in the protocol where it tells you to take small test samples at each step and run them on gels. They really do help you diagnose at what point you are going wrong.

Finally, I dont know if you are using the the newer high-speed kits, where you elute the DNA off a filter. I hate those, as the yields are always worse than the good old fashioned precipitation and centrifugation in the final two alcohol steps. AND do not overdry the DNA pellet at the end as it is a real problem to get it to completely redissolve once it has gone glassy (instead of white). Dissolving in pre-warmed Tris pH 8 is a good trick to make sure you get it all redissolved.

good luck!

Thanks so much for the advice. I talked with someone from Qiagen and turns out I have been overloading the columns 10-20 fold over the recommended amt of E. coli to load. Ouch. Also, I am using the leu2 yeast genomic libraries designed by Phil James. Does anyone know if these are high or low copy plasmids? The "HiSpeed" midi kit I am using elutes the DNA off of the filters. (Any potential problems here?) Thanks so much once again!