Gel Electrophoresis (2)
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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
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
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