If a bacteriophage A vector containing bacteriophageencoded RNA polymerase promoters was chosen as an expression vector

a. Generate a set of bacteriophage suspensions containing different ratios (10:1, 100:1, 1000:1, etc.) of an empty bacteriophage A. vector to a recombinant bacteriophage A. harboring the control cDNA. Infect an appropriate strain of E. coli with a multiplicity of bacteriophage particles that yields near-confluent lysis of bacterial lawns or complete lysis of infected cells grown in liquid culture (please see Chapter 2, Protocol 1 or Chapter 2, Protocol 5).

b. Prepare bacteriophage A, DNA from the plates (please see Chapter 2, Protocol 23) or from the liquid cultures (please see Chapter 2, Protocol 24).

c. Linearize the bacteriophage A. DNA at the rare restriction site placed at the 3' end of the cDNAs during library construction, and transcribe the cDNAs into mRNA as described in Chapter 9, Protocol 6.

d. Inject the prepared mRNA into Xenopus oocytes, and assay the mRNAs for their ability to encode the biological activity of the cDNA used as a positive control.

2. Using the results obtained in Step 1 as a rough guide, divide the cDNA library into pools of a suitable size for screening, and transform or transfect E. coli with the expression library pools. Prepare plasmid or bacteriophage A, DNA that will be used to screen the pools for the presence of the target cDNA.

Was this article helpful?

0 0
Caring For Your Lawn

Caring For Your Lawn

The Secret of A Great Lawn Without Needing a Professional You Can Do It And I Can Show You How. A Great Looking Lawn Doesn't Have To Cost Hundreds Of Dollars Or Require The Use Of A Professional Lawn Care Service. All You Need Is This Incredible Book.

Get My Free Ebook


Post a comment