Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(II) Principles of Biotechnology

Lecturer: Dr. Choo Quok Cheong

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Experiment No. 1

Title: Pipetting and Agarose Gel Electrophoresis

Date: 9 June 2009

Title: Pipetting and Agarose Gel Electrophoresis

Objectives:

– To learn the correct technique and precaution in using micropipette.

– To learn the methods and calculations involve in preparation of buffer solution.

– To know the components of different buffers and their functions.

 – To learn the methods in loading DNA into the well of agarose gel with

micropipette.

– To experience and carry out the agarose gel electrophoresis.

Materials and methods:

Refer to UESB 2142 Laboratory 2A Manual, page 12, 13, 14, and 15.

Questions:

1. (Weight of agarose powder/Volume of 1 x TAE buffer)/100% = 0.8%

(Weight of agarose powder/Volume of 1 x TAE buffer) = 0.008 g/ml

(Weight of agarose powder/20 ml) = 0.008 g/ml

Weight of agarose powder = 0.008 g/ml x 20 ml

= 0.16 g

2. (Weight of agarose powder/Volume of 1 x TBE buffer)/100% = 0.7%

(Weight of agarose powder/Volume of 1 x TBE buffer) = 0.007 g/ml

(Weight of agarose powder/50 ml) = 0.007 g/ml

Weight of agarose powder = 0.007 g/ml x 50 ml

= 0.35 g

3. Tris-Acetate-EDTA (EDTA) buffer is made up of Tris-acetate and EDTA, it is

usually at pH 8. TAE buffer has a lower ionic strength and a low buffering

capacity. TBE buffer contains borate instead of acetate. TBE buffer has high ionic

strength and buffering capacity.

TBE buffer has higher buffering capacity and ionic strength than TAE buffer, thus

TBE buffer offers higher resolution in electrophoresis in which DNA or RNA of

lower difference in kb can be differentiated. The borate in TBE is an inhibitor for

many enzymes such as Ligase, thus it can keep the DNA’s integrity better than
 TAE. However, TAE is more suitable if ligation is required in certain

electrophoresis.

4. Storage buffer and GTE buffer contain Tris-Cl buffer which is usually basic and

keep the DNA slightly basic and negatively charged so that DNA can be always

moves towards the anode. EDTA prevents the DNA or RNA from enzymatic

degradation as it chelate with metal ions that are usually cofactors to many

enzymes. The 1kb Blue DNA ladder is used as guideline in estimating the

unknown length of DNA samples in electrophoresis.

5. After the comb was being set on the agarose gel solution, the comb as well as the

gel cast should not be disturbed.

Before the power supply was turned on, the power cords should checked again if

it is already reversed.

Gloves should be worn when working with ethidium bromide.

The agarose gel should be viewed with UV transilluminator.

6. a) 200 ml 1 x TAE and 100 ml TE

200 ml 1 x TAE: M1V1 = M2V2

(50 x)(V1) = (1 x) (200ml)

V1 = (1 x) (200ml)/(50 x)

= 4 ml of 50 x TAE

Volume of distilled water required = 200 ml – 4 ml

= 196 ml

Therefore, 200 ml 1 x TAE buffer can be prepared by:

[4 ml of 50 x TAE] + [196 ml of distilled water]

100 ml TE: (10 mM Tris-Cl, 1 mM EDTA)

10 mM Tris-Cl: M1V1 = M2V2

(1 M)(V1) = (10 mM)(100 ml)

V1 = (10 mM)(100 ml)/(1 M)

= 1 ml of 1 M Tris-Cl

1 mM EDTA: M1V1 = M2V2

(0.5 M)(V1) = (1 mM)(100 ml)

V1 = (1 mM)(100 ml)/ (0.5 M)

= 0.2 ml of 0.5 M EDTA

Volume of distilled water required = 100 ml – 1 ml – 0.2 ml

= 98.8 ml

Therefore, 100 ml TE can be prepared by:

[1 ml of 1 M Tris-Cl] + [0.2 ml of 0.5 M EDTA] + [98.8 ml of distilled water]

b) 100 ml TE (pH 8) and 50 ml 5 x TAE

100 ml TE (pH 8): (10 mM Tris-Cl, 1 mM EDTA)

10 mM Tris-Cl: M1V1 = M2V2

(1 M)(V1) = (10 mM)(100 ml)

V1 = (10 mM)(100 ml)/(1 M)

= 1 ml of 1M Tris-Cl (pH 8)

1 mM EDTA (pH 8): M1V1 = M2V2

(1 M)(V1) = (1 mM)(100 ml)

V1 = (1 mM)(100 ml)/ (1 M)

= 0.2 ml of 0.5 M EDTA

Volume of distilled water required = 100 ml – 0.2 ml – 1 ml

 = 98.8 ml

Therefore, 100 ml TE (pH 8) can be prepared by:

[1 ml of 1M Tris-Cl (pH 8)] + [0.2 ml of 0.5 M EDTA] + [98.8 ml of distilled

water]

50 ml 5 x TAE: M1V1 = M2V2

(50 x)(V1) = (5 x)(50 ml)

V1 = (5 x)(50 ml)/(50 x)

= 5 ml of 50 x TAE

Volume of distilled water required = 50 ml – 5 ml

= 45 ml

Therefore, 50 ml 5 x TAE can be prepared by:

[5 ml of 50 x TAE] + [45 ml of distilled water]

c) 100 ml Solution I and 100 ml 1 x TAE

Solution I (25 mM Tris-Cl, 10 mM EDTA, 50 mM glucose)

25 mM Tris-Cl: M1V1 = M2V2

(1 M)(V1) = (25 mM)(100 ml)

V1 = (25 mM)(100 ml)/ (1 M)

= 2.5 ml of 1 M Tris-Cl

10 mM EDTA: M1V1 = M2V2

(0.5 M)(V1) = (10 mM)(100 ml)

V1 = (10 mM)(100 ml)/(0.5 M)

= 2 ml of 0.5 M EDTA

50 mM glucose: M1V1 = M2V2

(0.5 M)(V1) = (50 mM)(100 ml)

V1 = (50 mM)(100 ml)/(0.5 M)

 = 10 ml of 0.5 M glucose

Volume of distilled water required = 100 ml – 10 ml – 2 ml – 2.5 ml

= 85.5 ml

Therefore, 100 ml Solution I buffer can be prepared by:

[2.5 ml of 1 M Tris-Cl] + [2 ml of 0.5 M EDTA] + [10 ml of 0.5 M glucose] +

[85.5 ml of distilled water]

100 ml 1 x TAE: M1V1 = M2V2

(50 x)(V1) = (1 x )(100 ml)

V1 = (1 x)(100 ml)/(50 x)

= 2 ml of 50 x TAE

Volume of distilled water required = 100 ml – 2 ml

= 98 ml

Therefore, 100 ml 1 x TAE can be prepared by:

[2 ml of 50 x TAE] + [98 ml of distilled water]

d) 300 ml 1 x TAE and 100 ml Solution I

300 ml 1 x TAE: M1V1 = M2V2

(50 x)(V1) = (1 x )(300 ml)

V1 = (1 x)(300 ml)/(50 x)

= 6 ml of 50 x TAE

Volume of distilled water required = 300 ml – 6 ml

= 294 ml

Therefore, 300 ml 1 x TAE can be prepared by:

[6 ml of 50 x TAE] + [294 ml of distilled water]

Solution I (25 mM Tris-Cl, 10 mM EDTA, 50 mM glucose)

25 mM Tris-Cl: M1V1 = M2V2

 (1 M)(V1) = (25 mM)(100 ml)

V1 = (25 mM)(100 ml)/ (1 M)

= 2.5 ml of 1 M Tris-Cl

10 mM EDTA: M1V1 = M2V2

(0.5 M)(V1) = (10 mM)(100 ml)

V1 = (10 mM)(100 ml)/(0.5 M)

= 2 ml of 0.5 M EDTA

50 mM glucose: M1V1 = M2V2

(0.5 M)(V1) = (50 mM)(100 ml)

V1 = (50 mM)(100 ml)/(0.5 M)

= 10 ml of 0.5 M glucose

Volume of distilled water required = 100 ml – 10 ml – 2 ml – 2.5 ml

= 85.5 ml

Therefore, 100 ml Solution I buffer can be prepared by:

[2.5 ml of 1 M Tris-Cl] + [2 ml of 0.5 M EDTA] + [10 ml of 0.5 M glucose] +

[85.5 ml of distilled water]

References:

TAE buffer. (2009, May 17). In Wikipedia, the free encyclopedia. Retrieved June 13,

2009, from http://en.wikipedia.org/wiki/TAE_buffer

TE buffer. (2009, June 3). In Wikipedia, the free encyclopedia. Retrieved June 13, 2009,

from http://en.wikipedia.org/wiki/TE_buffer

TBE buffer. (2009, May 30). In Wikipedia, the free encyclopedia. Retrieved June 14,

2009, from http://en.wikipedia.org/wiki/TBE_buffer

DNA ladder. (2009, April 30). In Wikipedia, the free encyclopedia. Retrieved June 14,

2009, from http://en.wikipedia.org/wiki/DNA_ladder

EDTA. (2009, June 4). In Wikipedia, the free encyclopedia. Retrieved June 14, 2009,

from http://en.wikipedia.org/wiki/EDTA

About.com.(n.d.) How To Make TBE Buffer. Retrieved June 13, 2009, from

http://biotech.about.com/od/buffersandmedia/ht/MakeTBE.htm

About.com. (n.d.) How To Make TAE Buffer. Retrieved June 13, 2009, from

http://biotech.about.com/od/buffersandmedia/ht/TAE.htm

About.com. (n.d.) How To Make a Tris Buffer. Retrieved June 13, 2009,

from http://biotech.about.com/od/buffersandmedia/ht/trisbuffer.htm