The Amount Of Dna

Published Date: 02 Nov 2017

The amount of DNA in the sample was measured using the Qubit fluorometer (Qubit value = 5.4 μg / ml) and the concentration of the isolated plasmid DNA was calculated using equation 1.

Concentration of sample (µg/ml) = Qubit value (µg/ml) x (200/5) (equation 1)

Concentration of sample (μg/ml) = 5.4 μg/ml x (200/5)

Concentration of sample (μg/ml) = 216 μg/ml.

Agarose gel electrophoresis was used to separate DNA fragments and the presence of DNA bands was visualised (Picture 1) by capturing the image of the gel documentation system using computer software.

Picture 1: The presence of DNA bands

From left to right:

λ Hind III DNA marker (HindIII)

Undigested pAMP (C)

HindIII digested pAMP (B)

BamHI digested pAMP (H)

HindIII & BamHI digested pAMP (B/H)

7 bands of λ Hind III DNA marker were visualised and the distance migrated by fragment (mm) was measured using a ruler. Measurements are shown on table 1 below.

Table 1: Showing the distance migrated by fragment

Fragment number

Size of fragment (bp)

Log of Fragment size

Distance migrated by Fragment (mm)

1

23130

4.3642

15

2

9416

3.9739

18

3

6557

3.8167

21

4

4361

3.6396

26

5

2322

3.3659

37

6

2057

3.3132

41

7

564

2.7513

84

8

125

2.0969

-

Then using the information in the table 1 above, a calibration curve based on the bands produced by the λ HindIII DNA marker was plotted (Graph 1).

Graph 1: Calibration curve based on the bands produced by λ HindIII DNA size markers

Using the equation 2 (equation displayed on the Graph 1), the sizes of the unknown fragments were determined by inserting the distance migrated (mm). Data displayed on table 2 below.

y = - 0.0202 x + 4.3019 (equation 2)

Undigested pAMP: Distance migrated 20 mm (First band)

y = - 0.0202 x + 4.3019

y = - 0.0202 x (20) + 4.3019

y = 3.8979 (Log of fragment size)

So size of fragment (bp) = 7,904

Undigested pAMP: Distance migrated 37 mm (Second band)

y = - 0.0202 x + 4.3019

y = - 0.0202 x (37) + 4.3019

y = 3.5545 (Log of fragment size)

So size of fragment (bp) = 3,585

HindIII digested pAMP: Distance migrated 26 mm

y = - 0.0202 x + 4.3019

y = - 0.0202 x (26) + 4.3019

y = 3.7767 (Log of fragment size)

So size of fragment (bp) = 5,979

BamHI digested pAMP: Distance migrated 26 mm

y = - 0.0202 x + 4.3019

y = - 0.0202 x (26) + 4.3019

y = 3.7767 (Log of fragment size)

So size of fragment (bp) = 5,979

HindIII & BamHI digested pAMP: Distance migrated 28 mm (First band)

y = - 0.0202 x + 4.3019

y = - 0.0202 x (28) + 4.3019

y = 3.7363 (Log of fragment size)

So size of fragment (bp) = 5,448

HindIII & BamHI digested pAMP: Distance migrated 74 mm (Second band)

y = - 0.0202 x + 4.3019

y = - 0.0202 x (74) + 4.3019

y = 2.8071 (Log of fragment size)

So size of fragment (bp) = 641

Table 2: Showing the distance migrated and sizes of unknown fragments of pAMP

Fragment number

Unknown Fragment

Distance migrated by Fragment (mm)

Log of Fragment size

Size of fragment (bp)

1

Undigested pAMP (First band)

20

3.8979

7,904

2

Undigested pAMP (Second band)

37

3.5545

3,585

3

HindIII digested pAMP

26

3.7767

5,979

4

BamHI digested pAMP

26

3.7767

5,979

5

HindIII & BamHI digested pAMP (First band)

28

3.7363

5,448

6

HindIII & BamHI digested pAMP (Second band)

74

2.8071

641

12 petri dishes were prepared and the transformation reactions were preceded. After 24 hours the transformation plates were examined for presence of bacterial growth (Table 3)

Table 3: Showing the contents of each plate and whether or not bacterial growth resent

Plate Number

Plate Label

Sterile Water (ml)

Ampicillin 400µg/ml

(ml)

Kanamycin

400µg/ml

(ml)

Double Strength

NA (ml)

Mixture Added

Growth

1

C1

10

0

0

10

B

YES

2

C2

10

0

0

10

PA

YES

3

C3

10

0

0

10

CP

YES

4

C4

10

0

0

10

T

YES

5

A1

5

5

0

10

B

NO

6

A2

5

5

0

10

PA

YES

7

A3

5

5

0

10

CP

YES

8

A4

5

5

0

10

T

YES

9

AK1

0

5

5

10

B

NO

10

AK2

0

5

5

10

PA

NO

11

AK3

0

5

5

10

CP

YES

12

AK4

0

5

5

10

T

YES

B – Buffer

PA – pAMP plasmid

CP – Control plasmid

T – Transformation

Finally assuming that the transformation was undertaken with 0.1 μg of plasmid DNA (i.e. 0.1 μg in the 1 ml transformation reaction) the transformation efficiency was determined using equation 3.

Transformation efficiency (cfu / μg) = (Number of transformants x 20) / 0.1 (equation 3)

Growth greater than 300 cfu / ml was seen on plates, so using equation 3:

Transformation efficiency (cfu / μg) = (Number of transformants x 20) / 0.1

Transformation efficiency (cfu / μg) = (300 x 20) / 0.1 = 60,000 cfu / μg

Hence, transformation efficiency is greater than 60,000 cfu / μg.

Discussion

After bacterial transformation all 12 plates were examined for the presence of bacterial growth (Table 3). In plates C1, C2, C3 and C4 (Control plates) dense colonies (cfu > 300) of bacteria were observed. This was expected to happen as these plates were containing only E. coli and sterile water and no antibiotic was added. That observation confirmed the viability of bacteria and that transformation was successful.

On the other hand on the plate labelled A1 no bacterial presence was observed. In that plate E. coli was transformed with the buffer solution. Normally E. coli does not contain the Ampicillin resistance gene β-galactosidase, therefore untransformed bacterial were killed by the antibiotic, indicating E. coli sensitivity to Ampicillin.

On the plates A2, A3 and A4 containing pAMP plasmid DNA, control plasmid and transformation suspension respectively we had bacterial growth (less dense than C1 – C4). That was expected to happen as pAMP contains a gene that is resistant to Ampicillin allowing bacterial cells to grow. Moreover the presence of bacterial growth on plates A3 and A4 indicated that transformation was successful, however only with a number of set of bacteria being transformed.

The plates AK1 and AK2 displayed no growth as it was expected. The plate AK1 (with buffer) was containing both ampicillin and kanamycin. Therefore no growth of bacteria confirmed the sensitivity of untransformed E. coli to both of the antibiotics. The plate AK2 contained pAMP plasmid DNA (resistant to ampicillin) showed no growth indicating E. coli sensitivity to kanamycin.

Finally bacterial growth was displayed on the plates AK3 and AK4. On both plates only 16-17 colonies were present. Growth on plate AK4 was due to the presence of a gene on fragment F allowing kanamycin resistance.

According the results obtained, the transformation efficiency was greater than 60,000 cfu / μg. That means that indicated that a successful transformation of E. coli was carried out. Transformation efficiency can be affected by several factors such as the size of the colonies and the growth of the cells.