Biochem Report

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ladder.

Table 3: Shows the distance travelled by λ phage DNA (cm), the fragment size of λ phage DNA (kbp) and log10 (fragment size) of λ phage DNA.

Band

Distance travelled by the band (cm)

Fragment size (kbp)

Log10 (fragment size)

1

1.7

8.79

3.9441

2

2.9

3.39

3.5304

3

3.5

2.11

3.3236

4

3.9

1.53

3.1857

5

4.6

0.88

2.9444

[pic 5]

Figure 4: Shows the size of HindIII DNA ladder that should be expected from this experiment

Calculation

Example for band 1,

,[pic 6]

= x[pic 7]

This equation is obtained from figure 3.

Y = distance travelled by the bands

X = log10 (fragment size)

Therefore,

If Y= 1.7

X = [pic 8]

X = 3.9441

To know the fragment size (kbp);

Fragment size = 10x

Fragment size = 10 3.9441

Fragment size = 8792.25

= 8.79 kbp

Calculations are repeated for other fragments size of band 2, 3, 4 and 5 using the same methods.

Discussion

The size and distance pattern of migration for figure 1 matched with figure 2, as both of them have 15 bands of 1kb DNA ladder that appear in agarose gel as shown in figure 1. The HindIII sample which was digested with λ phage DNA shows only 5 bands sample instead of 8 band sample. According to, Restriction Digestion and Analysis of Lambda DNA Kit Instruction Manual, n.d 8 bands should have appeared on the agarose gel when HindIII cuts λ phage DNA. One of the reason why we only obtained 5 bands instead of 8 bands would be because the five fragments of HindIII digested by λ phage DNA sample was large enough to be noticed because it has adequate amount of DNA to be detected. Besides that, if the fragments migrate closely to each other on gel might affect the reading too and appear as a single band.

HindIII is a type of restriction enzyme that have four beta sheets and a single alpha helix and consist homodimer. The role of HindIII is it utilizes a common mechanism of recognition and catalysis of DNA found in other type II enzymes. These enzymes contain amino acid sequence that coordinate with Mg2+ and require DNA to be cleaved. The unique characteristics that HindIII holds is that it has little to no catalytic activity when magnesium ion is substituted for other cofactors.

There is a linear negative relationship between distances travelled by the DNA ladder to the fragment size. The distance travelled is inversely proportional to size of DNA fragments. So, the bigger the size of DNA fragments, shorter the distance travelled by each DNA fragments. The fragment size (kbp) for 5 bands of λ phage DNA was 8.79 kbp, 3.39 kbp, 2.11 kbp, 1.53 kbp and 0.88 kbp respectively but the expected fragment size for 8 bands would be 23.10 kbp, 9.42 kbp, 6.56 kbp, 4.36 kbp, 2.32 kbp, 2.03 kbp, 0.56 kbp and 0.13 kbp respectively. As a conclusion, it can be clearly seen that the experimental result is different from the expected HindIII digested with λ phage DNA.

Reference

Biswajit, M. and Basu, S. (2006). Fundamentals of Practical Clinical Biochemistry. New Delhi: B.I Publication Pvt Ltd, p.106.

Boyer, R. (2006). Biochemistry Laboratory : Modern Theory and Techniques. United State of America: Pearson Benjamin Cummings.

Deyl, Z., Everaerts, F. and Prusík, Z. (1979). Electrophoresis : A Survey of Techniques and Applications. Amsterdam [etc.]: Elsevier Scientific Publishing Co, pp.70-73.

Mackay, I., Volwiler, W., Goldsworthy, P., Eriksen, N. and Wood, P. (1954). PAPER ELECTROPHORESIS OF SERUM PROTEINS: PHOTOMETRIC QUANTITATION AND COMPARISON WITH FREE ELECTROPHORESIS 1. Journal of Clinical Investigation, 33(6), pp.855-866.

Restriction Digestion and Analysis of Lambda DNA Kit Instruction Manual. (n.d.). 1st ed. USA: Bio-Rad Laboratories, p.59

Switzer, R. and Garrity, L. (1999). Experimental biochemistry. New York: W.H. Freeman and Co.