7.3: Gene Amplification by Polymerase Chain Reaction (PCR)

I. Introduction:

In this experimeny, we are going to amplify E. coli tRNA2fMet(k12) gene by PCR technique. Two oligo DNA, NEWF2R and NEWF2L, are the primers. Both primers are 22 nucleotides long. Template DNA is pUC19/tRNA₂^fMet prepared in the experiment 9. The PCR product is a 220 bp fragment. Their corresponding sequences are shown in the introduction section.

II. Materials and Methods:

A. Materials:

1. Taq DNA polymerase: in 0.2 ml PCR tube	9. Marker B: pUC19/tRNA2fMet: Eco RI /HindⅢ digested (with gel loading dye)
2. 10x Taq polymerase buffer	10. 0.5x TBE buffer: 0.5 L
3. dNTPs mix	11. Thermocycler
4. Oligo primers	12. Gel electrophoresis apparatus
5. Template DNA	13. Microwave oven
6. RQ H2O	14. Balance
7. 6x agarose gel dye	15. Ink marker
8. Marker A : 100 bp markers

B. Procedure:

1. Set up PCR conditions.

2. Add into a 0.2 ml PCR tube the following reagents in the correct order:

a. PCR protocol:

Tube#	1	2	3
Component	reaction	Positive control	Negative control
1. double distilled water	18 ul	18	19
2.10 X PCR buffer	2.5 ul	2.5 ul	2.5 ul
3. dNTPs mix (2.5 mM/each)	2 ul	2	2
4. Primer L& Primer R mixture (100ng/ul)	1 ul	1	1
5. Taq DNA polymerase (5U/ul)	0.5 ul	0.5	0.5
6. Template (pUC19/tRNA2fMet) (1ng=E9 copies)	1 ul (template from your own preparation)	1 (template from TA)	0
Total vol. (l )	25 ul	25	25

b. PCR conditions:

	Temperature	Time (min:sec)	Cycle
Template denaturation	95	3:00	1
Template denaturation	95	0:30	25
Primer annealing	55	0:30
Enzyme extension	72	0:30
Final extension	72	3:00	1

3. Start the PCR reaction.

4. While PCR is running, prepare a 1.5% agar.

5. When PCR reaction is finished, mix with 5 ml of 6X agarose gel dye mix, load into a 1.5% agarose gel. A marker DNA (pUC19tRNA₂^fMet cut by Eco RI and HindⅢ) with dye is loaded on the gel side by side. Gel electrophoresis is set up at 100 volts for 30-45 min.

6. Marker A and B already contain gel dye, and add them into agarose gel well directly.

Marker A與B是有顏色的，不需再加入6x agarose gel dye，可以直接加入電泳膠片的樣品槽內。

Gel setting:

	lane 1	lane 2	lane 3	lane 4	lane 5
	Marker A (100 bp ladders)	Marker B (pUC19/fMet2 E/H cut	Reaction	Positive control	Negative control
DNA (ml)	20 (with dyes)	20 (with dyes)	25	25	25
6x agarose gel dye (ml)	0	0	5	5	5
Total vol. (ul)	20	20	30	30	30

7. Photograph of the gel results.

Note:

1. PCR caculation:

The average weight of a base pair (bp) is 650 Daltons.

The size of tRNA2^fMet gene is 220 bp.

The size of pUC19 plasmid is 2686 bp.

Template used in this PCR reaction is

1 ng DNA / ((220+2686)*650) = 5.3 x 10^-16 mole

5.3 x 10^-16 (mole) x 6.02 x10²³ = 3.2 x 10⁸ (template molecule number)

Nt = Nox2^cycle#

No = initial template input

Nt = final product output (amplicon)

IV. Questions:

1. Based on the OD260 reading of your own plasmid DNA preparation, how many copies of template you added into your PCR reaction? If the PCR efficiency is 100%, how many copies of PCR products can be obtained after 25 cycles?

2. 1 mg of E. coli genomic DNA equal to how many molecules? How about human cells?

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