Hi Alizadeh,

You have probably already solved your problem by now, however…..

If you want to produce helper phage you should make plaques and not colonies to obtain a monoclonal helper phage. Once the bacteria are infected the growth rate is significantly reduced. This is how the plaques are formed.

Here is the protocol I use, which gives me a good titre of helper phage (I use the KM13 helper phage).

Day 1

1: Make a 100-fold serial dilution of your helper phage (in 2xTY medium) and add 10 ul to 200ul fresh TG1 culture OD600 0.4. (I always use 2xTY when growing bacteria for phage infection/production).

2: Incubate at 37 for 30 min without shaking.

3: Add 3 ml molten top agar (42C) and pour onto warm LB (or TYE) plates. Allow top agar to set and incubate plates overnight at 37 degrees.

Note 1: I keep the top agar in 15 ml tubes in 42 degrees water bath and add the 210 ul (bacteria/phage mixture) directly to the top agar, give a mild vortex, and pour all on the plate. Be sure to distribute it evenly on the plate and try to avoid bubbles

Note 2: You can use TYE or LB agar mixed 1:1 with 2xTY or LB and use this as top agar.

Day 2

4: Next day you pick a small plaque into a fresh 5 ml TG1 culture OD600 of 0.4. Grow 2 hours shaking at 37 degrees (No antibiotics)

5: Transfer the 5 ml culture to a 2 litre flask with 500ml fresh 2xTY and grow 1 hour 37 degrees with shaking.

6: After the 1 hour you add the kanamycin (50ug/ml); NO glucose. Grow over night at 30 degrees. (I usually grow the culture 16-18 hours)

Day 3

7: Centrifuge the overnight phage culture. Transfer the supernatant to new tubes and add 20% PEG6000, 2.5M NaCl, 1:4, (10ml to 40ml supernatant). Store on ice for minimum 1 hour. You should see the mixture as slightly clouded.

Note: You can also use PEG8000. Both works well.

- Possible break – You can store the mixture overnight at 4C

8: Centrifuge 3,300xg for 45 min

9: Discard liquid and place tube upside down on paper towel to remove remaining PEG (10-15min)

10: Dissolve pellet in 10 ml PBS and centrifuge 3,300xg for 30 min to remove remaining bacteria

11: Transfer supernatant to a new tube and add 2.5 ml PEG solution. Place on ice for minimum 1 hour

12: Centrifuge 3,300xg 30 min.

13: Discard liquid and place tube upside down on paper towel to remove remaining PEG (10-15min)

14: Dissolve pellet in 5 ml PBS with 15% glycerol (glycerol is if you want to store the phage for long time, at -80C)

15: Finally, titre your helper phage

I usually get a titre higher than 10^13 pfu/ml following this protocol

I have an experience in working with M13 phage system in 2 scenarios(Peptide library and ScFv library).I used the same genetic system as mentioned by you and from my personal experience i would suggest you look at the following factors which ensures high phage titre.

(1)OD of the culture at the time of infection.Try to infect the cultures when OD is between 0.4 to 0.5.This is the most critical step when you miss this range you will have significant reduction in phage titre.

(2)Ensure that you are using the right PEG for phage precipitation,there are 2 versions PEG 6000 and 8000 you must use the 8000 version.

(3)Incubation of culture supernatant with PEG buffer should be strictly carried in cold conditions and optimizing the time of incubation will allow us to recover maximum Phage particles from culture supernatant.

Hope these suggestions help further you can look at this nature protocol which specific for Phage based ScFv library screening.

You might also try a richer media or buffered media. I have found that I get higher titers using Terrific Broth instead of 2xYT.

Terrific Broth

12g Bacto tryptone

24g Bacto yeast extract

4ml glycerol

Add H2O to 900ml and autoclave, then add to above sterile solution 100ml of a sterile solution of 0.17M KH2PO4 and 0.72M K2HPO4.

Dear Alireza

I am working on Lytic phages and As my experiences there is some notices:

1. The media that recruited for bacteria culture; It must be 2x TSB or LB broth.

2. Use overnight (fresh) bacteria

2. The OD of host; It must be 1.5-3 macfarland.

3. Increase the volume; Use 50 ml of Host.

4. The incubation time must not be over than 6hrs.

5. Add 1-2 ml Mg2So4 (10%) to the bacterial suspension.

Don't vortex or shake suspension firmly. Physically forces facilitate Pilli lose.

Hi Alizadeh,

You have probably already solved your problem by now, however…..

If you want to produce helper phage you should make plaques and not colonies to obtain a monoclonal helper phage. Once the bacteria are infected the growth rate is significantly reduced. This is how the plaques are formed.

Here is the protocol I use, which gives me a good titre of helper phage (I use the KM13 helper phage).

Day 1

1: Make a 100-fold serial dilution of your helper phage (in 2xTY medium) and add 10 ul to 200ul fresh TG1 culture OD600 0.4. (I always use 2xTY when growing bacteria for phage infection/production).

2: Incubate at 37 for 30 min without shaking.

3: Add 3 ml molten top agar (42C) and pour onto warm LB (or TYE) plates. Allow top agar to set and incubate plates overnight at 37 degrees.

Note 1: I keep the top agar in 15 ml tubes in 42 degrees water bath and add the 210 ul (bacteria/phage mixture) directly to the top agar, give a mild vortex, and pour all on the plate. Be sure to distribute it evenly on the plate and try to avoid bubbles

Note 2: You can use TYE or LB agar mixed 1:1 with 2xTY or LB and use this as top agar.

Day 2

4: Next day you pick a small plaque into a fresh 5 ml TG1 culture OD600 of 0.4. Grow 2 hours shaking at 37 degrees (No antibiotics)

5: Transfer the 5 ml culture to a 2 litre flask with 500ml fresh 2xTY and grow 1 hour 37 degrees with shaking.

6: After the 1 hour you add the kanamycin (50ug/ml); NO glucose. Grow over night at 30 degrees. (I usually grow the culture 16-18 hours)

Day 3

7: Centrifuge the overnight phage culture. Transfer the supernatant to new tubes and add 20% PEG6000, 2.5M NaCl, 1:4, (10ml to 40ml supernatant). Store on ice for minimum 1 hour. You should see the mixture as slightly clouded.

Note: You can also use PEG8000. Both works well.

- Possible break – You can store the mixture overnight at 4C

8: Centrifuge 3,300xg for 45 min

9: Discard liquid and place tube upside down on paper towel to remove remaining PEG (10-15min)

10: Dissolve pellet in 10 ml PBS and centrifuge 3,300xg for 30 min to remove remaining bacteria

11: Transfer supernatant to a new tube and add 2.5 ml PEG solution. Place on ice for minimum 1 hour

12: Centrifuge 3,300xg 30 min.

13: Discard liquid and place tube upside down on paper towel to remove remaining PEG (10-15min)

14: Dissolve pellet in 5 ml PBS with 15% glycerol (glycerol is if you want to store the phage for long time, at -80C)

15: Finally, titre your helper phage

I usually get a titre higher than 10^13 pfu/ml following this protocol

Morten Dræby Sørensen , can we use M13KO7 helper phage in place of KM13?

Both are same or different, do you have any idea ? please tell me

You can amplify twice. If you get low titer initially, amplify those again to maximize the amplification titer.