Enclosed you will find a cDNA library made in the lambda vector lYES-R or ACT (activation domain) and several other strains depending upon your request. The titer should be 1 x 109 PFU/ml or greater, with between 1 x 107 and 1 x 108 total recombinants, amplified only once from the packaging. Upon reciept, these phage should be titered, amplified to give a higher titer stock if necessary, and aliquotes can be frozen (9% DMSO, -70°C) for future use including DNA preparation. Libraries should be titered and amplified as phage on LE392 or equivalent bacterial host (NOT BNN132).
The cDNA was sized selected to be >600bp. The bacterial strain is BNN132 = JM107/lKC, a kanr lambda lysogen containing the cre gene. Infection of this strain with the library will produce Ampr colonies that have quantitatively excised the plasmid via cre-mediated lox recombination. I typically infect the library into log phase JM107/lKC cells and plate about 108 infected cells (108 phage in 3 x 108 cells) per large LB Amp-50 ug/ml plate (add 0.2% glucose if using lYES-R). I usually absorb the phage for 30′ at 30°C and then add LB and allow the cells plus phage to grow for an hour before plating. This allows the cells to express the bla gene and probably to undergo recombination. Since lYES-R was designed to express both in E. coli and yeast, I use the glucose to help repress the lac promoter (JM107 is also lacIq). This helps prevent biasing of the library. lACT does not contain the lac promoter so glucose is not required. I usually plate about 10 large plates worth of library which is about 100 times the original number of recombinants. I have found no difference between plating at 30°C or 37°C. The lKC phage has a wild type repressor. DNA can be prepared directly from the lysogens scraped from these plates. I usually take these cells and resuspend them in terrific broth and grow them up a little further to get more DNA (6 liters gives 2 mg of plasmid DNA). Remember, these are pBR322 based plasmids so the copy number is not high. There is no real reason not to do the whole thing in liquid. I think that growth on plates presents less of a competitive situation for the clones, but it may not really be needed in actuality. For further information I suggest you read PNAS 88:1731-1734 on the lYES-R system. Two other publications concerning the 2-hybrid system are Genes and Dev. 7:555-569. and JBC 268:4608-4611.
Although this goes without saying, these libraries and strains should not be distributed to other researchers without my prior consent and should be used for only the experiments you described in your letter. Good luck.
Conversion of a library in l ACT into a plasmid library
* Logarithmically growing BNN132 bacteria
* 10 fresh 150 mm LB, ampicillin (50 mg/ml), 0.2% glucose plates
* 3 liters of terrific broth (16)
1. Mix 108 phage from the amplified library with 2 ml of logarithmically growing BNN132 bacteria( 3 x 108cells/ml) + 10mM MgCl2.and incubate at 30°C without shaking for 30 minutes. The number of phage and cells used can be scaled-up for better representation.
2. Add 2 ml of LB and shake at 30°C for 1 h., cells can be concentrated or plated directly.
3. Spread 200 ml of the cells on each of 10 fresh, 150mm LB/50mg/ml ampicillin/ 0.2% glucose plates and incubate overnight at 37°C. You should see a lawn of cells. Occasionally small plaques are seen in the lawn. These can be safely ignored. Dilutions of the original infection should be plated to determine excision efficiency. Poor efficiency is usually due to using stationary phase BNN132 cells, too much ampicillin in the plates, or no MgCl2 added during absorption.
4. Add 10 mls of LB to each plate and resuspend the ampicillin-resistant colonies. Pool the bacteria from all 10 or more plates and use to inoculate 3 liters of “terrific broth” (16) with 50 mg/ml ampicillin. Grow to stationary phase and make plasmid DNA by CsCl gradients. 3 liters of cells will produce 1 or 2 mg plasmid DNA.
This can be scaled down for individual phage to plasmid conversion. It is advisable to amplify the cDNA library as phage prior to conversion to plasmid. This ensures that you will have plenty of opportunities to convert to plasmid form now or in the future.