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How to calculate the size of DNA fragments using unknown marker length

The size of DNA fragment or the gene of interest could be estimated using standard markers (like 1kb, 100bp markers etc.). If our fragment run between the known marker fragment, then this method is useful.

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How to calculate the size of DNA fragments using unknown marker length

1. 1. Calculating the size of DNA fragments using unknown marker length Nishanth Sekar PhD (Ag) in Microbiolgy
2. 2. Problem: Calculate the size of A, B, C using the Hind III digest of lamba DNA marker Create standard curve using DNA marker Measure distance travelled by unknown DNA fragment Determine size of DNA fragment Size of unknown A, B, C ? !
3. 3. Create standard curve using DNA marker Size of DNA fragment in bp log 10 Distance travelled by the fragments (cms) 23130 4.3642 4.4 9416 3.9739 5.0 6557 3.8167 5.9 4361 3.6396 7.1 2322 3.3659 9.2 2027 3.3069 9.8 564 2.7513 14.8 125 2.0969 17.5
4. 4. Measure distance travelled by unknown DNA fragment Distance travelled by A = 6.5 cm Distance travelled by B = 10.7 cm Distance travelled by C = 13.7 cm
5. 5. Determine size of DNA fragment A B 6.5 cm 10.7 cm 13.7 cm 2.7 3.2 3.8 A : antilog (3.8) = ~ 6309 bp B : antilog (3.2) = ~ 1585 bp C : antilog (2.7) = ~ 501 bp C
6. 6. Using MS Excel Simple linear regression equation y = mx+c In this case… y = -0.149x+4.792 Size of DNA fragment in bp log 10 Distance travelled by the fragments (cms) 23130 4.3642 4.4 9416 3.9739 5.0 6557 3.8167 5.9 4361 3.6396 7.1 2322 3.3659 9.2 2027 3.3069 9.8 564 2.7513 14.8 125 2.0969 17.5 Distance travelled (cms) Value of y Antilog A 6.5 3.8235 6660.395 B 10.7 3.1977 1576.522 C 13.7 2.7507 563.2483 Trendline !!! Size of A, B and C