Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.10 T7 phage viability assay 3

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Small Phage March - April Notebook: Experiments

4.10 T7 Phage Viability Test 3

I) Purpose

- Test the viability (concentration if possible) of the T7 (new) and T7+ we got

- Become familiar with virus tittering techniques

II) Expected outcome

- Should see multiple plaques forming on a lawn of E coli (confluent growth)

- Plaques should decrease in size as the dilution gets smaller

III) Reagent record

T7 phage + and T7 new ; E coli BL21; Agar: ×2 prepared by Jordan in 500mL glass bottle; LB: prepared by Jordan in big Erlenmeyer flask; overnight bacteria culture: set up on Tues 4/9 at 12pm

IV) Actual procedures / observations

1) Dilution series of bacteriophage and its incubation with E coli

-For T7 (new)

i) 90μL of LB broth were added to added to each dilution vial, labeled 1-15.

ii) 10μL stock phage solution (T7 new)was added to vial 1.

iii) Perform a serial dilution (1:10) with vial 1-5, decreasing phage concentration to 10% of the previous vial with each.

-For T7+

i) 90μL of LB broth were added to added to each dilution vial, labeled 6-15.

ii) 10μL from tube 5 of T7+ dilution series (4.8 T7 Phage Viability Test 2) was added to vial 1.

iii) Perform a serial dilution (1:10) with vial 6-15, decreasing phage concentration to 10% of the previous vial

with each.

2) Perform spot test

i) In 50mL plastic centrifuge tube, combine 2mL of E. coli BL21 overnight liquid culture, 10mL of warm ×2 top agar, and 10mL LB.

ii) In another 50mL plastic centrifuge tube, combine 1.5mL of E. coli BL21 overnight liquid culture, 7.5mL of warm ×2 top agar, and 7.5mL LB (sup).

iii) 5mL of the combined solution were added to each LB plate. Wait for top agar to solidify before proceeding to spot test.

iv) Divide each plate into 6 sections. For T7 (new), they were labeled 0-5, 5-10, and 10-15. For T7+, they were labled 5-10 and 10-15. Spot 5μL of liquid from each vial onto different sections of the plate. Vial and section should correspond in number. On section 0, 5μL taken from phage liquid stock were spotted directly (performed for both T7+ and T7 (new) individually).

v) Set up one E coli control plate by taking 5mL of the combined solution from the centrifuge tube (2-i step) and plated it on an LB plate.

3. Phage plaque size test

i) Add 20 μL of T7+ phage from centrifuge tubes 5, 10, and 15 to glass test tube labelled 5, 10, and 15, respectively

ii) Add 0.5mL E. coli BL21 from overnight liquid culture to test tube

iii) Let both test tube sit for 20min

iv)In a 50mL centrifuge tube, combine 10mL of warm ×2 top agar and 10mL of LB to produce ×1 top agar

v) Add 5mL of ×1 top agar to each test tube, pipet up and down several times before plating 5mL unto LB plates

4) Check up on phage + bacteria viability in 24 hours.

IV) Results

1) Plates

Both spot tests for T7+ and T7 (new) formed plaques up to section 6. Sections 7-15 had no plaques.

T7 (new) spot tests

ADD PICTURE

T7+ spot test

ADD PICTURE

2) Only the -5 titer formed plaques.

Titer Test

ADD PICTURE

V) Conclusions

- T7 (new) is able to grow on E coli BL21.

- Plaques decrease in size from sections 0-6 in a 1:10 dilution series. After that, no plaques form

- T7+ titer of -5 formed many individual plaques, while -10 and -15 didn’t form any.

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-| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Small Phage May - June Notebook: Experiments'''</font>
+| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Small Phage March - April Notebook: Experiments'''</font>
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