Screening lambda cDNA or genomic libraries

Media

Need LB plates (9 cm or 14 cm)to plate the library and do secondary screens.I typically screen about 500,000-1 million plaques for a primary cDNA screen.This fits on 11-22 14 cm plates.I do the secondary screens on 9 cm plates- you should pour about 20 of these so you will be prepared in you get a bunch of positives.After pouring the plates,let them age/dry at room temperature for 2-3 days.This is necessary to help the top agarose stick.You will also need LB top agarose containing 10 mM MgCl2 or MgSO4.I use 0.9% agarose ("low electroendo-osmosis" agarose is best,it is slightly low melting.If regular agarose is used,you may need to go down to 0.7% to get the top agarose to spread evenly before it sets.)

Note: many protocols call for using special "NZY" plates for plating lambda libraries.In practice,LB medium works fine.

Bacterial host strain

- For most lambda strains use RY1073 or C600 HflA or LE392 for lambda hosts.Actually almost any e coli (like TG1)are ok for some lamda vectors,like lambda ZAP II.For lambda gt11 use the host Y1090.The Barstead mixed stage C.elegans cDNA library is in the original lamda ZAP vector,and requires a host carrying the supF mutation,such as Y1090.

Growing the cells:

grow up 40 ml of bacteria o/n in LB

spin 5 min,5K

resuspend in 10 mM MgCl2 (1/2 original volume)

these "2X cells" can be kept in the fridge & used for ~1 month

Note: optionally,can add maltose to 0.2% to the LB medium used to grow the cells.Make a sterile filtered stock of 10% maltose to supplement the LB.Maltose induces higher expression of the lambda receptor.In practice,the screening will work fine even if you don't add maltose to the medium.

Titering the library

Libraries are stored at 4℃ in a tightly sealed tube,with a ｄｒｏｐ of chloroform at the bottom (chloroform keeps bacteria from growing but won't harm the phage).For very long term storage,add 10% DMSO,freeze in liquid nitrogen,store at -70°.Titers will range from 106-1011 phage per ml.Often a 1000-fold diluted stock of the original library is titered and stored for use.Further dilutions made to use for a particular experiment are discarded after the experiment is done- these more dilute phage solutions tend not to hold their titer.

Phage will be diluted in 1X phage buffer.Here is the recipe for 10X phage buffer:

10X phage buffer

100 mM Tris pH7.5

100 mM MgCl2

500 mM NaCl

Prepare some top agarose:

Melt a bottle of top agarose in the microwave.For 100 mm plates,put 3 mls in each of several 13X100 mm glass culture tubes in a 50-55° temp block.For 15 cm plates need 6.6 ml top agarose (use a bigger tube and a 50-55° water bath).

to titer (on 100 mm plates):

Put various dilutions of phage in 100 µl 1X phage buffer.Always do a no phage control! This will help you distinguish between dilutions that give you no phage (look like the no phage control)and dilutions that give complete lysis (too many phage so that no individual plaques can be distinguished).

To 100 µl diluted phage

add 100 µl 2x cells

incubate 37° for 20'

plate with 3 ml top agarose (on a 100 mm plate)

To plate,squirt the phage/cell mixture into a tube,vortex quickly,pour onto a 100 mmlB plate,quickly tilt the plate around to evenly coat the plate with the top agarose.Do not use LB plates straight out fo the cold room- they will cause the top agarose to set before you can spread it evenly.Let the plates sit a few minutes for the top agarose to harden.Then turn the plates upside down and move to 37°.

grow 37° for 6-8 hours for cDNA library,~10 hrs for a genomic library.This should be enough time to get nice clear ~1 mm plaques.

You want the library to be plated for screening at a density such that the plaques can grow to about 1 mm in diameter before they begin to just barely touch each other.For your first screen,get an experienced person to look at your titering plates to give you advice about what dilution to use.If you are going to titer on 10 cm plates and plate on 15 cm plates,you will have to multiply everything by 2.25 to adjust to the large plates.