In labeled eppendorf tubes, prepare the dilutions indicated in the table below (
ID: 190883 • Letter: I
Question
In labeled eppendorf tubes, prepare the dilutions indicated in the table below (use calculations from pre-lab protocol). Prepare 100 ul of the most concentrated dilution (using non-boiled samples), and then prepare 50 ul of the other dilutions from that most concentrated dilution (dilute in Tris-PMSF buffer). Keep these samples on ice 5. Dilution Volume of sample (indicate if sample was re-diluted) (ul 50 Volume Tris-PMSF (ul) Total volume (ul) 100 50 50 50 Crude homogenate 1:2 Crude homogenate 1:5 Crude homogenate 1:10 Crude homogenate 1:20 AMS resuspended 1:2 AMS resuspended 1:5 AMS resuspended 1:10 AMS resuspended 1:20 AMS desalted 1:2 AMS desalted 1:5 AMS desalted 1:10 AMS desalted 1:20 NADH undiluted NADH 1:2 NADH 1:4 5 (1:2 dil)Explanation / Answer
Dilution
Volume of Sample (indicate if the sample was prediluted) (ul)
Volume Tris-PMSF (ul)
Total volume (ul)
Crude homogenate (1:2)
50
50
100
Crude homogenate (1:5)
20 (1:2 dil)
30
50
Crude homogenate (1:10)
10 (1:2 dil)
40
50
Crude homogenate (1:20)
5 (1:2 dil)
45
50
AMS resuspended (1:2)
50
50
100
AMS resuspended (1:5)
20 (1:2 dil)
30
50
AMS resuspended (1:10)
10 (1:2 dil)
40
50
AMS resuspended (1:20)
5 (1:2 dil)
45
50
AMS desalted (1:2)
50
50
100
AMS desalted (1:5)
20 (1:2 dil)
30
50
AMS desalted (1:10)
10 (1:2 dil)
40
50
AMS desalted (1:20)
5 (1:2 dil)
45
50
NADH undiluted
100
0
100
NADH (!:2)
25 (undiluted)
25
50
NADH(1:4)
12.5 (undiluted)
37.5
50
Dilution factor= Final Volume/Initial volume
For 1: 2 Dilution,
Dilution factor=50/Initial volume
Initial volume=50/dilution factor= 50/2= 25ul
When using prediluted samples for dilution:
a) !: 5 dilution from 1: 2 dilution, the dilution becomes 1:2.5
Hence, add 10 ul for total volume of 25 ul. This becomes 20 ul for total volume of 50 ul.
20 ul of 1: 2 diluted sample + 30 ul of Tris-PMSF.
b) !: 10 dilution from 1: 2 dilution, the dilution becomes 1:5
Hence, add 5 ul for total volume of 25 ul. This becomes 10 ul for total volume of 50 ul.
10 ul of 1: 2 diluted sample + 40 ul of Tris-PMSF.
c) !: 20 dilution from 1: 2 dilution, the dilution becomes 1:10
Hence, add 2.5 ul for total volume of 25 ul. This becomes 5 ul for total volume of 50 ul.
5 ul of 1: 2 diluted sample + 45 ul of Tris-PMSF.
Query: What is * in NADH undiluted. If * is the most concentrated solution, then use the above calculation with undiluted sample. If not, use 1:2 dilution as most concentrated solution to calculate 1:4 dilution. In that case, it will be 25ul of 1:2 dilution +25ul of Tris-PMSF= 50ul total volume.
Dilution
Volume of Sample (indicate if the sample was prediluted) (ul)
Volume Tris-PMSF (ul)
Total volume (ul)
Crude homogenate (1:2)
50
50
100
Crude homogenate (1:5)
20 (1:2 dil)
30
50
Crude homogenate (1:10)
10 (1:2 dil)
40
50
Crude homogenate (1:20)
5 (1:2 dil)
45
50
AMS resuspended (1:2)
50
50
100
AMS resuspended (1:5)
20 (1:2 dil)
30
50
AMS resuspended (1:10)
10 (1:2 dil)
40
50
AMS resuspended (1:20)
5 (1:2 dil)
45
50
AMS desalted (1:2)
50
50
100
AMS desalted (1:5)
20 (1:2 dil)
30
50
AMS desalted (1:10)
10 (1:2 dil)
40
50
AMS desalted (1:20)
5 (1:2 dil)
45
50
NADH undiluted
100
0
100
NADH (!:2)
25 (undiluted)
25
50
NADH(1:4)
12.5 (undiluted)
37.5
50
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