Nov 23, 2020
P1 transduction
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. P1 transduction. protocols.io https://protocols.io/view/p1-transduction-bpznmp5e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 23, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44814
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Prepare P1 phage
Prepare P1 phage
Inoculate a single colony into 5 ml LB medium (+ appropriate antibiotic if needed)
Shake at 37 °C overnight
Dilute to be 0.01 in 10 ml LB medium + 50 µL of 1 M CaCl2 + 0.2% glucose final
Grow at 37 °C for 60 minutes (don’t want the cells to get much above 0.1 OD600)
Add 10 µL , 50 µL , or 100 µL P1KC phage lysate
Grow at 37 °C until lysed (ideally~3 hr)
Add 200 µL drops chloroform, vortex for 30 seconds
Spin at ? for 10 minutes
Transfer the supernatant into a new 15 ml tube
Add 100 ul chloroform
Store in fridge
P1 transduction
P1 transduction
Inoculate a single colony into 5 ml LB medium (+ appropriate antibiotic, if needed)
Shake at 37 °C overnight
Spin down cells
Resuspend in 5 ml MC buffer (10 mM MgCl2, 5 mM CaCl2)
Set the transduction mix in a 1.5 ml Eppendorf tube
Tube#1 | Cells | P1 lysate
1 0.1 ml _
2 0.1 ml 10 ul
3 0.1 ml 50 ul
4 0.1 ml 100 ul
5 - 100 ul
Incubate at RT for 20 min without shaking
Add 20 µL 1 M NaCritrate
Shake at 37 °C for 1 hr
Plate 100 µL on LB plate + appropriate antibiotic
Incubate plate at 37 °C overnight
Examine colonies = transductants