PCR assembly reaction

Summary

This experiment describes about the process of PCR assembly reaction. This experiment is from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

PCR assembly reaction

Materials and Instruments

Magnesium chloride Rockstart buffer three-reaction master mix Agarose gel
PCR Instrument PCR Tubes

Move

I. Materials

1. Buffers, solutions and reagents

Magnesium chloride, 35 mmol/L

Rockstart buffer (http://klentaq.com)

three-reaction master mix

82ul 5mol/L betaine

3.2ul dNTP mixture at 10 mmol/L each

1ul Klentaq LA enzyme mixture (Clontech 8421-1)

6.3ul Left PCR riboprimer (storage solution concentration 10 umol/L)

6.3ul Right PCR riboprimer (storage solution concentration 10umol/L) (no template)

153.5ul _H20 Make up to total volume 252ul

2. gel

Agarose Gel, 1.4%

3. special equipment

PCR instrument

PCR tubes

II.

1. 10ul of Rockstart buffer and 10ul of 35 mmol/L MgCl2 were added to each of the 3 PCR tubes, and the components were allowed to stand and mix for at least 15 min to form a magnesium precipitate (Barnes and Rowlyk 2002).

2. Prepare the three-reaction main harbor composite.

3. After formation of the iron precipitate, add 80ul of the three-reaction master mix to each reaction tube containing Rockstart precipitate.

4. add 1ul or 2ul of 40mer to the mixture at a concentration of lpmol/ul of each 40mer. it is not important whether the mixture is mixed or not. Perform 25 cycles at 93°C for 1 min and 62°C for 20 min.

5. 15ul of PCR product was analyzed by agarose clot electrophoresis at 1.4% agarose.

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