The following protocols outline some of the methods we have developed for picking, PCR and sequencing of large numbers of cDNA clones. With any cDNA library the titre, percentage of non-recombinants and range of insert sizes should be know before proceeding on a larger scale.

Plating out, picking and archiving clones from cDNA libraires.

PCR amplification of clones and size selection for sequencing.

Regridding of PCR products for sequencing size selected clones

SAP/EXO I cleaning of PCR products and sequencing

Recovery of phage from DMSO stocks

*NOTE* all our protocols are designed to process clones in a 96 well microtitre plate format. The use of multichannel pippettes1 is strongly reccommended.

AMPLIFICATION, PICKING AND ARCHIVING OF CLONES FROM cDNA LIBRARIES

Day 1 -Prepare overnight culture of XL1-Blue cells

XL1-Blue cells should be maintained on LB tet agar 2,3plates (4°C) and subbed regularly to fresh plates.

1 Pick a single colony of XL1-Blue into 50ml NZYDT4 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.

3. These cells are now ready for infection with phage proceed to step 4 or store cells at 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

Per 90mm NZY agar plate you will require:

200ul XL1-Blue in 10mM MgSO4 (OD600nm=0.6 )

small volume of cDNA library to ensure 50-100 pfu per plate

3mls NZY top agarose

50ul 250mg/ml X-Gal

15ul 0.5M IPTG

Prepare sufficient

 

NZY5 select agar plates (Freshly prepared plates should be dried for 30-60 mins at 37°C to prevent streaking).

NZY top agarose6 (3mls required per 90mm plate).

You should know the titre of your phage stock (plaque forming units (pfu) per ml). Aim to plate out approximately 50-100 pfu per plate. This should ensure the plaques are well separated and easy to pick. Picking and archiving 1000-1500 plaques (10-15 microtitre plates) is a good number for one person to pick in a day

4. Put NZY plates into dry/prewarm (30-60 min) ready for plating in step 9.

5. Melt NZY top agarose and equilibrate to 50°C in waterbath ready for plating in step 9.

6. Prepare aliqouts of XL1-Blue cells for infection. Per 90mm agar plate you require 200ul of cells. Add the correct volume of cDNA library to the cell alliquots to ensure 50-100pfu per 200ul of cells.

eg if titre of library =1x 106 pfu/ml = 1x103 pfu/ul.

Mix phage and host cells well.

7. Incubate for 20 min at 37°C to allow phage to attach to cells.

8. If the library has a high percentage of non recominants use blue/white colour selection to ensure only recombinant phage are picked. Add the X-Gal7 and IPTG8 to the top agarose.

The X-Gal and IPTG must be added to the top agarose only once it has equilibrated to 50oC. The X-Gal may precipitate when added to the top agarose. Try to disperse it by mixing well. The precipitate does not usually affect the blue/white selection or the growth of cells.

9. Add 3mls NZY top agarose 50oC to each 200ul aliquot of infected cells, mix and plate 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 .

10. Invert plates and incubate overnight at 37°C.

Day 3- Picking phage to microtitre plates and making DMSO archive

The plaques should be picked a soon as possible to prevent the possibility of cross contamination due to phage diffusion. Only well separated plaques should be picked. If blue/white clour selection has been used only white (clear plaques) should be picked.

V bottomed well plates9 are better for standing tips in and gilson pipette tip box insert placed onto of the microtitre plate helps to support the tips and makes it easier to remove them (see Figure 1)

Figure 1

Prepare sufficient Microtitre plates labelled in duplicate:

One set of plates will be used for primary picking of phage and should be prepared with 25 ul Phage Storage Buffer (PSB)10 per well.

The replicate set of plates will be used for archiving the phage and should be prepared with 5 ul 20% DMSO11 per well.

1. Using sterile yellow tips core the centre of the plaques and transfer the tips to individual wells of a microtitre plate containing 25 ul PSB. (A small volume of agarose/fluid should picked up by the tip).

2. Leave the tip standing in the well for approximately 20 min to allow the phage to diffuse into the PSB (Tips are typically left in one plate while the next is picked).

3. Carefully remove the tips from the plate taking care not to cross contaminate wells. This is the primary pick plate.

4. 10 ul of the 25 ul from the primary pick plate should then be transferred to the replicate plate containing 5 ul 20% DMSO. Mix well. (This is the archive plate containing 15 ul phage in PSB 7% DMSO)

5. Archive plates should be frozen -80°C as soon as possible. The phage remain viable in DMSO but the plates should not be freeze thawed as this will reduced the viability of the phage.

6. The remaining 15 ul phage/PSB of the primary pick is used for PCR/sequencing and can be freeze thawed.

PCR protocol

Notes

Archive plates should be kept on dry ice if taken out of -80°C

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 or streaking across a lawn of XL1-Blue in top agarose. Phage rescue protocol

Scrapes from the DMSO stock can also be used to PCR the clone inserts .

Footnotes

1 Autoclavable multichannel pipettes from Annachem cat no:

2. LB AGAR (PER LITRE)

OR

LB mix can be bought from GIBCOBRL cat no. 12795-027

3. Tetracycline (tet) 12 mg/ml

4. NZYDT broth (per litre)

OR

NZYDT broth mix can be bought from-GibcoBRL cat no. 23650-021

5. 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

6. NZY top agarose

OR

NZY mix can be bought from-GibcoBRL cat no 13635-024

7. X-GAL (5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside) Boehringer cat no 10011 dissolved in DMF (N,N-dimethyl Formamide) Sigma cat no D8654.

8. IPTG (Isopropyl-B-D-Thiogalactopyranoside) Boehringer cat no 10085

9. V well microtitre plates and lids Grenier cat no 651161 and 656171.

10. PSB (sterile)

11. DMSO (dimethylsulfoxide) Sigma cat no. D8418