due 24 hours

Today’s Lab

Welcome!

Experiment II: Day 7 October 4, 2016

•  Count the pfu for each plate

The plaques are round areas of clearing in the solid lawn of host bacteria (E. coli KL227 in this instance)

•  Using the dilution that you trust most to back-calculate the concentration in your P1Cm c1-100 tube from 4°C

•  If your phage titer is low, please let us know and we will give you the P1Cm c1-100 culture for today

–  You want to add 1x108 phage (pfu/ml) to your tube

rifR (rpoB) Mutant

200 µl

P1Cm c1-100

NO PHAGE

108 pfu 1ml

X pfu = ml to add

100mM CaCl2

5mM

C1V1 = C2V2

Incubate at 30°C for 10’

ADD PHAGE

To determine CaCl2:

Calculate how much phage to add:

Calculation of number of phage particles per ml

Phage/ml = [(number plaques/plate) x

dilution factor] / ml plated e.g. if you got 350 plaques when you plated out

0.2 ml of a 106 diluted phage suspension, the titer for your phage is: 1.75 x 109/ml

350 pfu x 106 / 0.2 ml = 1.75 x 109 pfu/ml

•  In meantime prep two minimal media plates: spread 100µl each trp, his, and met

•  After incubation add sodium citrate to TUBES a final concentration (f.c.) of 10mM

•  Centrifuge for 20s **BALANCE the tubes and put on the LID**

•  Discard supernatant

•  Resuspend in remaining liquid

•  Spread on plates

•  Incubate at 30°C(& NOT 37°C. Correct this on your lab manual please)

Clean-Up •  Used…

– amino acids – Salt – citrate – white freezer boxes – tips

•  …go on cart in back

•  Old plates & tubes to autoclave waste bins

•  New plates at 30°C