Table of Contents
- 1 What does chloroform do to phages?
- 2 What is used for the preparation of phage lysate?
- 3 How do you dilute phage?
- 4 What is the use of chloroform to prepare lysate during phage isolation?
- 5 How do you make phage stock?
- 6 What is high titer lysate?
- 7 What is SM buffer?
- 8 What is the role of chloroform in isolation of Coliphage experiment?
- 9 Can you add chloroform to phage prep?
- 10 Can a chloroform centrifuge be used on phage?
- 11 Which is the best way to store phage?
What does chloroform do to phages?
If you add a drop of chloroform to the phage prep and shake it around that will kill many bacteria. Then you can add the phage prep to your indicator bacteria. But chloroform also kills the indicator bacteria so you have to be careful.
What is used for the preparation of phage lysate?
Protocol for Phage Lysate Preparation Prepare a fresh plating of phage using rich media (M9supp plates and top agar are optimum media, but phage plates and soft agar can be used, or even LB). Transfer 1 ml of overnight culture to 4 ml of rich media in a 50-ml flask (M9supp is optimum media, but LB will also work).
How do you increase phage in titre?
For this, you can add 100 microlitres from your purified phage stock to your grown host culture (3 to 5 ml). Incubate overnight. Centrifuge the suspension to pellet the bacteria, collect the supernatant and filter sterilize it. This would usually enrich initial phage stock to 100-1000 times.
How do you dilute phage?
Prepare serial 1/10 or 1/100 dilutions of the phage stock using 0.9 ml or 0.99 ml SM blanks. The first dilution should be a 1/100 dilution to minimize the volume of the original phage stock used. Dilutions in the range expected to produce valid plate counts should be 1/10 dilutions.
What is the use of chloroform to prepare lysate during phage isolation?
Chloroform is normally added to the phage isolation/purification step to get rid of bacteria. Afterwards it should be removed (e.g. by centrifugation) before using the phage preparation. Chloroform is not an essential step and it should be taken into account that it may inactivate other phages present in the bacteria.
What is a high titre phage?
Phage titer is expressed in PFU (Plaque Forming Units)/mL. Lysates with a final concentration greater than 109 PFU/ml are “High Titer” lysates. While the best countable plate has between 20 – 200 plaques, dilution techniques must be verified by observing a 10-fold decrease in plaque numbers across the dilutions.
How do you make phage stock?
Phage stock preparation
- Grow E.
- Prepare and autoclave 1 Molarity (M) CaCl2 and 1 Molarity (M) MgCl2.
- Incubate with agitation for 01:00:00 at 37 °C .
- Add 100 µl of high titer phage lysate (> 108 PFU/ml).
- Incubate at 37 °C with agitation for ∼ 05:00:00 or until lysate clears.
What is high titer lysate?
A plate lysate is simply a concentrated liquid sample of phage. High titer phage lysates will yield sufficient quantities of DNA for sequencing. Plate lysates are the standard for long‐term storage of a phage sample.
How do you calculate phage stock?
For example, if the plate you selected was the 10^-5 plate, you would multiply 1570 by 10^5 to get 157000000. This final number is your phage titer, and represents the number of viruses per ml of your original culture.
What is SM buffer?
Introduction: SM Buffer is a mixture of Sodium chloride, Magnesium sulphate and gelatin and is extensively used in molecular biology. This solution is a sterile-filtered, pre-mixed solution. This is used as a phage diluent and storage buffer as the gelatin stabilizes lambda phage.
What is the role of chloroform in isolation of Coliphage experiment?
Chloroform was added to remove bacterial remnants and a virion suspension was carefully recovered. Lysis tests and bacteriophage counts were further performed by plate assays.
Does chloroform cause cell lysis?
The chloroform is added for 15-20 min to lyse the cells thus releasing the virus particles to the medium.
Can you add chloroform to phage prep?
If you add a drop of chloroform to the phage prep and shake it around that will kill many bacteria. Then you can add the phage prep to your indicator bacteria. But chloroform also kills the indicator bacteria so you have to be careful. Chloroform is much heavier than water.
Can a chloroform centrifuge be used on phage?
Many but not all phage are not harmed by chloroform. As an alternative you can centrifuge and/or filter away bacteria. If there are not enough bacteria to make a mess on your indicator lawn there may not be a need to do any bacterial removal at all.
How are phage stocks prepared in bacterial culture?
Preparation of phage stocks is a vital step in Phage biology. In a bacterial culture, the phage suspension multiplies by taking bacterial cell machinery and lyse the cells eventually by releasing new phage particles. As a result, the medium contains a high concentration of phage particles.
Which is the best way to store phage?
The most applicable method for long term storage of phage stock is placing them in SM buffer at 4 o C. SM buffer or SM phage diluents which contains gelatin, Chloroform and Mg 2+ are used for routine manipulation of phage stocks. The gelatin used in SM buffer helps to stabilize the phage particles while storage.