Cell Bio help due sunday!! NO time to complete because of Lab
final please please help
Harvesting cells

What I need to turn in

*A table with concentrations and absorbencies

*The graph of the standard curve with the tren line, y=
equation, r squared= value

*the ug/ul of the sample

*the ul needed for 30ug of the sample

*the amounth of 4x dye needed for the sample

Experiment: Harvesting cells

1)Remove media by dumping into bleach, 2)wash the plate twice
with 2mL of 1XPBS, 3)add 500uL of 1XPBS and scrape the plate for
cells, 4)transfer the liuquid w the scraped cells into a conical
tube, repeat steps 3&4 with 500uL of 1XPBS and then 1mL of
1XPBS, Spin the conical tube with media and cells at 400G or
1950rpm for 4minutes, aspirate or pour the supernattant from the
conical tube and transfer the pellet into eppendorf tube by adding
500uL of PBS into the conical tube, then transfer the liquid to an
eppendorg tube. Be care not to dislodge the pellet when aspirating,
spin the eppendrf tube at 400G or 1950rpm for 4 minutes, Remove the
PBS, leaving the pellet, lyse the cells- KEEP ON ICE, make a RIPA
buffer mix of 100uL RIPA buffer and 1uL protease, add aproximately
80uL RIPA buffer mix to the pellet, vortex the pellet with the RIPA
buffer mix, leave in ice for 15 minutes vortexing every 5 minutes,
spin at 10,000 RPM for 10 minutes in refrigerated centrifuge

Bradford Assay method

a standard curve must be created in order to have a compare
unknown concentrations to known concentrations,

1) obtain 7 curvettes and lable them B, B, 1,2,4, 8, S 2) The B
curvettes will not have any BSA since it serves as a control sample
to calibrate the B spectrophotometer 3) “1” add 1uL of BSA to the
bottom corners 4) “2” add 2uL of BSA 5) “4” add 4uL of BSA 6) “8”
add 8uL of BSA 7)”S” add 2uL of your sample you harvested 8)add1mL
of biorad to each curvette 9) measure the abosorbance using the
spectophotometer Creating a standard curve & calculating
unknown concentrations   

to create a calibration curve you will use the absorbencies you
measure for the BSA concentrations of 1, 2, 4 and 8

1) to creat the curve, plot the known values in excel as a
scatter plot, 2) Once you have th graph go to layout-trend line-
add linear treandline. 3) double click on the trend line and select
“display equation on chart: and display r squared value you will
see y=#x+# and r squared=# pop up on your graph. 4) to determine
your unknown concentrations a) divide the abosorbance of your
sample by the value next to the x in they=equations {EX. if
y=.0354x+44, you do y-44 divided by .0354} b) find houw many ug per
uL by dividing the concentration by 2 c) find uL needed for 30ug by
dividing 30 by the ug per uL 5) find how much due needed by
dividing the uL needed for 30uG by 4

After lab was completed absorbance values were

BSA 1uL .075

BSA 2uL .162

BSA 4uL .330

BSA 8uL .505

Sample 2uL .453