|
|
|
|
Prepare a culture of XLI-Blue cells and infect the cells with phage from your cDNA library to determine titre of your stock.
Day 1 -Prepare overnight culture of XL1-Blue cells
XL1-Blue cells should be maintained on LB tet agar 1,2 plates (4°C) and subbed to fresh plates weekly.
1 Pick a single colony of XL1-Blue into 50ml NZYDT3 broth. Grow cells overnight with shaking at 37°C.
Day 2-Preparation of XL1-Blue
1. Pellet 50ml overnight culture (1000 x g for 15 min) and gently resuspend cells in small volume of sterile 10mM MgSO4 .Do not vortex.
2. Further dilute an aliquot of the resuspended cells from step 1 using sterile 10mM MgSO4 to OD600nm=0.6. Prepare a sufficient volume of cells for the number of platings required.
These cells are now ready for infection with phage.
3. Proceed to step 4 or store cells 4°C till use. (Best titre results are obtained with freshly prepared cells but cells can be stored for up to one week at 4°C)
Infection of XL1-Blue and plating of phage
Prepare
NZY4 select agar plates (Freshly prepared plates should be dried for 30-60 mins 37°C to prevent streaking). NZY top agarose5 (3mls required per 90mm plate).
4. Put NZY plates into dry/prewarm (30-60 min) ready for plating in step 8.
5. Melt NZY top agarose and equilibrate to 50°C in waterbath ready for plating in step 8.
6 Take a small aliquot of your library (eg 5ul) and prepare serial diltuions of your library in PSB ranging from 10-2 to 10-7. You now have 6 working dilutions of your library (This range of diltuions should be sufficient to titre most libraries).
6. Prepare 12 aliquots of 200ul XL1-Blue cells (from step 2) in 7ml bijous
7 Add 2, and 10ul of each library dilution to seperate aliquots of 200ul XL1-Blue cells. You now have 12 seperate infections of cells with different volumes of cDNA library.
7. Incubate cells and phage for 20 min at 37°C to allow phage to attach to cells.
8 While cells are incubating prepare the top agarose . You require 3mls top agarose per plate . To determine the percentage of non recombinants you need to use blue/white colour selection. For this you need to add 50ul 250mg/ml X-Gal, 15ul 0.5M IPTG to each 3ml top agarose.
For 12 plates prepare
The X-Gal and IPTG must be added to the top agarose only once it has equilibrated to 50 C. The X-Gal may precipitate when added to the top agarose. This does not usually affect the blue whit selection or the growth of cells.
8. Add 3mls NZY top agarose 50oC to each aliquot of infected cells, mix and pour out onto prewarmed NZY plates. Gently swirl the NZY top agarose around the plates before it sets to ensure even distribution of plaques (nb. NZY top agarose sets at 42°C). Allow NZY top agarose to set .
9. Invert plates and incubate overnight at 37°C.
Day 3 - You should expect to see well seperated plaques on at least some of your plates. If not you may need to try other diltuions of phage stock.
Some of the plaques may appear blue. Blue plaques are produced by non recombinant phage. Clear plaques are produced by recombinant phage containing inserts. Where possible you should count the number of plaques present on each plate noting the percentage of plaques that are blue. From the number of plaques on each plate and the volume of library diltion used to infect the cells on the plate you can determine the titre and percentage of non recominants in your library.
You may want to PCR some randomly selected clones and PCR the inserts to check the quality and range of inserts in your library. To do this you need to pick individual clones from your plates and PCR from them.
Select a plate from your titration that has well separated plaques. The plaques should be picked a soon as possible to prevent the possibility of cross contamination due to phage diffusion.
1. Using sterile yellow tips core the centre of 20 plaques. A small volume of agarose/fluid should picked up by the tip. Transfer the tip to a sterile eppendorff containing 25 ul PSB.
2. Allow the phage to diffuse into the PSB for approx 20 min at room temp.
3. Remove the tips and mix well to ensure dispersal of phage. This is now a PSB stock of the clone and can be used for PCR.
PCR protocol
Notes
The phage/PSB stock can be stored at 4oC for several months if chloroform is added (1:50). This stock can be used as a viable source of phage. If the phage/PSB stock is frozen at anytime the phage will be unlikely to be viable on thawing and will only be useful as a template for PCR.
A frozen archive of clones can be made by transferred 10ul of the 25ul phage/PSB stock to a fresh tube containing 5ul of 20% DMSO (final conc 7% DMSO) and mix well. Freeze this DMSO stock immediately at -80oC. This is now a DMSO stock of an individual clone. This should not be done if Chlorofrm has already been added.
Phage can be rescued from the DMSO stocks by taking a scrape from the frozen well using a sterile needle and inoculating into 200ul XL1-Blue (as in step 6) or streaking across a lawn of XL1-Blue in top agarose.
Scrapes from the DMSO stock can also be used to PCR the clone inserts .
Footnotes
1. LB AGAR (PER LITRE)
OR
LB mix can be bought from GIBCOBRL cat no. 12795-027
2. Tetracycline (tet) 12 mg/ml
3. NZYDT broth (per litre)
OR
NZYDT broth mix can be bought from-GibcoBRL cat no. 23650-021
4. NZY agar per litre
OR
NZY mix can be bought from-GibcoBRL cat no 13635-024
Select agar can be bought from Gibco BRL cat no 30391.023
5. NZY top agarose
OR
NZY mix can be bought from-GibcoBRL cat no 13635-024
6. PSB (sterile)
7. DMSO (dimethylsulfoxide) Sigma cat no. Sigma D8418