Team:Queens Canada/error2

Error Prone PCR

Introduction

Error prone PCR is a method by which random mutants may be inserted into any piece of DNA.

Materials

• • DNA primers for PCR amplification
  • DNA template
  • Four separate solutions of 20 mM dATP, TTP, dCTP, and dGTP
  • Taq DNA polymerase, stored at -20 C
  • 10 X PCR buffer: 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, and 500 mM KCl
  • 1 MgCl2
  • 50 mM MnCl2
  • Agarose
  • 10 X TBE buffer: 1 M Tris base, 1 M boric acid, and 20 mM EDTA, pH 8.0
  • 10 mg/mL ethidium bromide
  • Loading buffer: 10 mM Tris-HCl, pH 7.6, 0.15 % orange G, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA
  • DNA mass ladder for quantification
  • Gel purification kit

Procedure

1. Prepare a stock solution of the DNA template (~50-100 ng/μL) in water.

2. Label 16 thin-walled PCR tubes as "reactions 1 through 16".

3. Combine the reagents listed in Table 1 in a conical vial and label it "PCR reaction mixture"

4. Dispense the PCR reaction mixture from step 3 into tubes 1 through 16 by 96 μL to tube 1 and add 88 μL to tubes 2-16.

5. Add 2 μL of the DNA template to tube 1.

6. Place tube 1 in the thermocycler and start the PCR program (Table 2).

	A A	B B	C C	D D
1 1	Reagents	Stock Concentration	Volume	Final Concentration
2 2	Forward Primer	100 μM	15 μL	1 μM
3 3	Reverse Primer	100 μM	15 μL	1 μM
4 4	dCTP and dTTP	20 mM	75 μL/ea	1 mM
5 5	dATP and dGTP	20 mM	15 μL/ea	0.2 mM
6 6	PCR buffer w/ Mg2+	10X	150 μL	1x
7 7	MgCl2	1M	8 μL	~5.5 mM
8 8	DMDC H2O		1098 μL
9 9	Final Volume		1491 μL

PCR Reagent Mixture

	A A	B B	C C	D D
1 1	Step	Temperature	Duration
2 2	Template Denaturation	95 - 98°C	1 min	One time only
3 3	Oligo Denaturation	94°C	1 min	Repeat next 3 steps x30
4 4	Annealing	58°C	1 min	This may change depending on primers
5 5	Extension	72°C	2 min
6 6	Final Elongation	72°C	5 min
7 7	Product Storage	4°C	forever	Repeat steps 3-5 for 30 cycles

Table1

7. Once the PCR program has reached the annealing temperature, add 1 μL of freshly prepared MnCl2 solution and 1 μL of Taq DNA polymerase to the PCR reaction tube.

8. Perform 4 cycles of PCR amplification using the hot start procedure (see Note 3).

9. Remove the PCR tube from the thermocycler

10. Place the tube on ice.

11. Transfer 10 μL of the PCR reaction from tube 1 to tube 2.

12. Place tube 2 in the thermocycler and start the PCR program.

13. Once the PCR program has reached the annealing temperature, add 1 μL of freshly prepared MnCl2 and 1 μL of Taq DNA polymerase to the PCR reaction tube.

14. Perform 4 cycles of PCR amplification using the host start procedure.

15. Remove the PCR tube from the thermocycler

16. Place the tube on ice.

17. Repeat steps 11 through 16 using tubes 3 through 16 to create a mutagenic library by serial dilution amplification (image down below)

18. Verify the quality of each PCR reaction by agarose gel electrophoresis.

19. Combine 50 μL of each PCR reaction into a single tube and store on ice; this is your DNA library

20. Purify the DNA library by agarose gel electrophoresis

21. Recover the DNA library using a gel purification kit.

22. Quantify the DNA library by agarose gel electrophoresis

• NOTES

23. Primer should be complemetary and have similar melting techniques: http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm

24. Optimal PCR conditions should be determined prior to the use of this mutagenic PCR protocol. The doubling efficiency for the normal PCR reaction should be ∼1.7–1.9 per PCR cycle and can be analyzed by running the PCR product after each cycle on an agarose gel

25. Taq DNA polymerase should be stored at −20°C and kept on ice or in a frozen metal block when taken out of the freezer. The hot start procedure refers to adding the Taq DNA polymerase to the PCR reaction mixture after the mixture has reached the annealing temperature. Either standard or “hot start” Taq DNA polymerase will work in the described method.

26. It is important to prepare a fresh MnCl₂ solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.

27. It is important to prepare a fresh MnCl₂ solution for the error-prone PCR experiment. This solution should be stored on ice and combined with the PCR reaction mixture at the start of each serial dilution.

28. The method described here was optimized for a 400-nucleotide gene. We highly recommend that experimenters read ref. 7 for information on the predicted average number of doublings and mutations expected as a function of template length

29. Agarose gel electrophoresis should be performed by standard methods. A DNA ladder with an appropriate base pair range should be used to identify and estimate the quantity of the PCR product.

30. It is recommended to save a portion of the PCR product from each tube for further analysis or amplification.

31. The concentration of the DNA product can be determined by comparing the intensity of the product bands to the intensity of the bands from the DNA quantification ladder from commercial sources.

• Transformation of Libraries

32. GIbson Assembely can be performed if the primers of the linearized vector are complimentery to the error-prone PCR product.

References

1. Random Mutagenesis by Error-Prone PCR McCullum E Williams B Zhang J Chaput J Methods in molecular biology (Clifton, N.J.) 2010 vol: 634 pp: 103-109