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generated protocol from Brigid's creamatocrit SOP
==Mouse Milk Creamatocrit (milk fat %)==
===Materials===
*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)
===Materials===
*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)
