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Electrophoresis

Essay by   •  April 19, 2017  •  Lab Report  •  1,318 Words (6 Pages)  •  609 Views

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Lab report 3: Electrophoresis

Results

The following table and the graph give the results obtained when the distance travelled by the fragments of the DNA marker was approximately measured using a ruler along with the corresponding fragment size

[pic 1]

Figure 1: UV image resulting from the agarose gel electrophoresis

Table 1: fragment size (bp) and corresponding distance (cm) travelled by fragments observed for the sample.

Band number

band size (bp)

Log(band size)

Distance travelled (cm)

1

20000

4.30

1.5

2

10000

4.00

1.6

3

7000

3.84

1.7

4

5000

3.70

1.9

5

4000

3.60

2.0

6

3000

3.48

2.3

7

2000

3.30

2.7

8

1500

3.18

3.0

9

1000

3.00

3.8

10

700

2.85

4.4

11

500

2.70

4.8

12

400

2.60

5.1

13

300

2.48

5.5

14

200

2.30

6.0

15

75

1.88

6.6

[pic 2]

Figure 2: graph of log (fragment)/bp and distance travelled for fermentas ladder

Table 2: Distance travelled by the fragments resulting from Hind III digestion of the λ phage DNA and their calculated sizes

Band number

Distance migrated/cm

Calculated size/bp

1

2.5

3162

2

3.0

1995

3

3.5

1259

4

4.2

794

5

Not visible

Was not visible to calculate

6

Not visible

Was not visible to calculate

7

Not visible

Was not visible to calculate

8

Not visible

Was not visible to calculate

Calculations:

y = -2.710x + 11.97

Where y= distance travelled and x = log (fragment size)

When y= 2.5

2.5 = -2.710x + 11.97

X= 3.5

Log x= 3.5

103.5= X

Fragment size=3162

When y= 3.0

3.0 = -2.710x + 11.97

X=3.3

Log x= 3.3

X= 103.3

Fragment size=1995

When y= 3.5

3.5= -2.710x + 11.97

X=3.1

Log x= 3.1

X= 103.1

Fragment size=1259

When y= 4.2

4.2 = -2.710x + 11.97

X=2.9

Log x= 2.9

X= 102.9

Fragment size=794

Discussion

The results obtained from this experiment are very flawed even though the graph shows a very strong correlation value of 0.977. Some bands were not visible and some appeared like large smears. The bands of the DNA marker used (Gene ruler 1kb Plus DNA ladder, SM1333) and that of the sample were not the same number as expected. Many errors could have caused such erroneous results.

According to the fragmentation diagrams of the DNA marker and the restriction enzyme obtained from the Fermentas Website, the DNA ladder should be separated into 15 bands from which only 14 were observed in the UV image obtained after electrophoresis and only 4 bands (4 fragments) were observed for that of the HindIII marker (eight were expected). This could have happened due to incomplete digestion of the DNA sequence by restriction endonucleases. (Agarwal & Singh, 2010). Another reason that less than 8 bands appeared is because the buffer pH was not effective. The pH of buffer is a major factor which determines the migrated distance of sample bands. (Smith, 1996). Also, the gel might have not been stained for enough time as small fragment size would need more time to be stained because they are in lower concentration than heavy bands. Some of the sample in the wells had not migrated even slightly. It could be because the sample was not pipetted into the well properly and could have diffused into the buffer.  (Hirano, 2010). The first two bands (20000 and 10000 bp) got mixed into 1 band. Bands having fragment sizes of 4000, 1500 and 500 bp were easily identified on the gel image as they appear brighter.

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