Reference no: EM133009534
Part A: Denaturation and Tm Determination
Methods:
This first component of the practical has been done for you. Six samples of dsDNA were heated in a water bath form a starting temperature of 37.5°C to 92°C in 2.5°C increments. The absorbance at 260nm was recorded for each sample at each temperature point. The data collected is represented in Table 1. Within the set of samples there is a range of fragment lengths, salt concentrations and G/C content.
Table 1: Spectrophotometric analysis of dsDNA samples
|
|
Abs @ 260 nm
|
|
Temp oC
|
Sample
1
|
Sample
2
|
Sample
3
|
Sample
4
|
Sample
5
|
Sample
6
|
|
37.5
|
0.49
|
0.51
|
0.80
|
0.79
|
0.50
|
0.49
|
|
40
|
0.50
|
0.50
|
0.80
|
0.80
|
0.50
|
0.49
|
|
42.5
|
0.49
|
0.49
|
0.80
|
0.80
|
0.49
|
0.50
|
|
45
|
0.5
|
0.5
|
0.80
|
0.80
|
0.50
|
0.49
|
|
47.5
|
0.50
|
0.5
|
0.80
|
0.80
|
0.49
|
0.50
|
|
50
|
0.50
|
0.50
|
0.80
|
0.79
|
0.50
|
0.50
|
|
52.5
|
0.52
|
0.49
|
0.79
|
0.80
|
0.50
|
0.50
|
|
55
|
0.54
|
0.50
|
0.80
|
0.80
|
0.51
|
0.50
|
|
57.5
|
0.59
|
0.50
|
0.80
|
0.80
|
0.53
|
0.49
|
|
60
|
0.63
|
0.50
|
0.80
|
0.79
|
0.61
|
0.50
|
|
62.5
|
0.65
|
0.51
|
0.81
|
0.80
|
0.67
|
0.50
|
|
65
|
0.66
|
0.54
|
0.84
|
0.80
|
0.67
|
0.50
|
|
67.5
|
0.67
|
0.60
|
0.91
|
0.80
|
0.67
|
0.52
|
|
70
|
0.67
|
0.66
|
0.97
|
0.80
|
0.67
|
0.62
|
|
72.5
|
0.68
|
0.68
|
1.02
|
0.80
|
0.68
|
0.67
|
|
75
|
0.68
|
0.68
|
1.07
|
0.82
|
0.67
|
0.67
|
|
77.5
|
0.67
|
0.67
|
1.08
|
0.88
|
0.67
|
0.67
|
|
80
|
0.68
|
0.68
|
1.07
|
0.98
|
0.67
|
0.67
|
|
82.5
|
0.67
|
0.67
|
1.08
|
1.05
|
0.67
|
0.67
|
|
85
|
0.67
|
0.67
|
1.08
|
1.07
|
0.67
|
0.67
|
|
87.5
|
0.67
|
0.67
|
1.08
|
1.08
|
0.68
|
0.68
|
|
90
|
0.68
|
0.68
|
1.08
|
1.08
|
0.68
|
0.68
|
|
92.5
|
0.68
|
0.68
|
1.08
|
1.08
|
0.68
|
0.68
|
Part A:
Graph all six samples with the temperature in °C on the X axis and Abs260 on the Y axis. You should plot two samples per graph, samples 1 & 2 on one, 3 & 4 on a second and 5 & 6 on the third. For each sample you will need to determine the Tm by picking the corresponding temperature for the mid-point between the lowest and highest Abs260.
Q1: What shape does the melting curve have, is there a linear relationship between denaturation and temperature?
Q2: For each graph, the only difference between the two samples was the salt concentration. Has this produced a difference in Tm and how much for each pair?
Q3: Why does salt concentration affect Tm?
Part 2:
There are a number of different methods you can use to calculate the Tm oligonucleotide fragments. In the introduction to the practical three were mentioned, the 2+4 method, a slightly more complicated calculation which takes into account salt, size and %G/C factors and the nearest neighbour method. Use the formula below to calculate the Tm for each DNA sample listed in Table 2.
Tm = 81.5 + 16.6(log10 [Na]) + 0.41(%G/C) - 600/oligo length
Table 2: Experimental conditions for DNA samples
|
Sample #
|
Length (bp)
|
[Na M]
|
%G/C
|
|
1
|
25
|
0.05
|
51
|
|
2
|
25
|
0.2
|
51
|
|
3
|
50
|
0.05
|
51
|
|
4
|
50
|
0.2
|
51
|
|
5
|
25
|
0.05
|
57
|
|
6
|
25
|
0.2
|
57
|
Q4: Samples 1&2 differ from 5&6 in base composition, what effect did this have on Tm and did your calculated values agree with the differences observed on the graphs?
Use the 2 + 4 method (2°C for every A/T pair and 4°C for every G/C pair to determine Tm for sample 5. Does this correlate well with the calculated value from the equation above?
Q5: Why would you not use the 2 + 4 method to calculate Tm of longer oligos (e.g. 50 bp) and use the method to indicate why?
Q6: Samples 3 and 4 had higher Abs260 than the other four samples, why do you think this is the case?
Attachment:- Denaturation and Tm Determination.rar