Difference between revisions of "Milk Creamatocrit"
From Bridges Lab Protocols
(generated protocol from Brigid's creamatocrit SOP) |
(added more to materials) |
||
| Line 1: | Line 1: | ||
==Mouse Milk Creamatocrit (milk fat %)== | ==Mouse Milk Creamatocrit (milk fat %)== | ||
===Materials=== | ===Materials=== | ||
| + | *Milk samples (from -80) | ||
| + | *Ice bucket | ||
*CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower) | *CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower) | ||
*1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA) | *1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA) | ||
| − | * | + | *Untreated glass hematocrit tubes |
*Fractional dial calipers | *Fractional dial calipers | ||
*Critoseal | *Critoseal | ||
Revision as of 15:43, 17 March 2022
Contents
Mouse Milk Creamatocrit (milk fat %)
Materials
- Milk samples (from -80)
- Ice bucket
- CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower)
- 1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA)
- Untreated glass hematocrit tubes
- Fractional dial calipers
- Critoseal
- Kimwipes
- P10 pipette with tips
Preparing the samples
- Thaw whole milk on ice. To homogenize thawed milk, pipette up and down for five seconds. DO NOT shake!
- Dilute the milk in aa 1:3 solution with the 1XPBS + EDTA. Make enough for 4 capillary tubes
- Take capillary tube and draw in enough milk to fill ~3/4 of the tube (stop before the blue line)
- Hold horizontally and seal the other end of the tube with critoseal twice. Be sure to continue to hold the tube horizontally, or the milk will be ejected.
- Wipe excess milk from the tube with the kimwipe
- Each sample should be run in duplicate (you may need to run additional tubes if the variance between the first 2 sample results is >=25%)
Running samples
- Place sealed and filled capillaries into the critspin micro-hematocrit spinner. The sealed end should point toward the outer edge of the centrifuge.
- Record the position of each tube/sample before running..
- Screw on the centrifuge cover so that it is secure, but not too difficult to loosen later.
- Spin for 120 seconds, eight separate times. Between spins, the centrifuge will come to a complete stop on its own.
- After the 8th spin, immediately read the creamatocrit using the calipers
Reading the samples
- Measure to the nearest 0.01 inch the aqueous layer, the fat layer, and the small aqueous layer on top of the fat layer.
- Record results in a csv file.
- Calculate the milk fat percentage using the following R code:
mutated.milk.data<-milk.data%>%
group_by(replicate, MouseID, Genotype)%>%
mutate(fat = Fat.inches*25.4,
water = (Aqueous.inches.1 + Aqueous.inches.2)*25.4)%>%
mutate(water.corrected = water/4)%>%
mutate(total.volume = water.corrected+fat)%>%
mutate(fat.percent = (fat/total.volume)*100)
