Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period2/Exp/4.8 T7 phage viability assay 2
From 2013.igem.org
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- Test the viability (concentration if possible) of the T7 we got | - Test the viability (concentration if possible) of the T7 we got | ||
- | - Become familiar with virus | + | - Become familiar with virus titering techniques |
- Identifying T7 mutant and T7 WT | - Identifying T7 mutant and T7 WT |
Revision as of 22:35, 5 June 2013
Small Phage May - June Notebook: Experiments
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4.8 T7 Phage Viability Test 2
I) Purpose - Test the viability (concentration if possible) of the T7 we got - Become familiar with virus titering techniques - Identifying T7 mutant and T7 WT II) Expected outcome - Should see multiple plaques forming on a lawn of E coli (confluent growth) - T7 WT should grow, even though T7 mutant didn’t (4.5 phage viability assay) III) Reagent record T7 phage *; 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 Sat 3/6 at 6pm IV) Actual procedures / observations 1) Dilution series of bacteriophage and its incubation with E coli
2) Perform spot test
30 Phage plaque size test
4) Check up on phage + bacteria viability in 24 hours. IV) Results 1) Plates
2) Spot test compared to control (top: BL21; bottom: W3110) ADD PICTURE Circular plaques formed for each spot test. The plaques decreased in size as phage in LB becomes more diluted. No contamination seen. 3) Titer test compared to control (top: BL21; bottom: W3110) ADD PICTURE Phage is clearing up the entire plate. This indicates that the phage we used are at too high of a concentration. V) Conclusions - T7* is the WT T7 phage. It is able to grow on E coli BL21. - Our stock phage liquid culture is at too high concentration to form a few individual plaques on a plate. It’s clearing up the entire area. VI) Proposed next step 1) Perform the same spot test with more diluted T7 solution. Look for where the plaques are barely forming.
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