( A high concentration results in a reduc...)Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

A high concentration results in a reduction in specificity and incurs unnecessary costs.

1234L Figure 3: The effect of DNA polymerase amount on PCR specificity Figure 3 demonstrates that 0.250.5 U enzyme are often enough sufficient for a 50 l preparation and that with high concentrations of enzyme nonspecific by-products are increasingly synthesized.

Amplification of a DNA fragment (length 117 base pairs) on a plasmid (10.4 kbp) using a standard protocol. Every parameter - except the amount of Taq polymerase - was maintained at a constant level. Volume 50 l. 1: Taq polymerase: 0.25 U
2: Taq polymerase: 0.5 U
3: Taq polymerase: 1 U
4: Taq polymerase: 2 U
L: 100-bp ladder

Buffers and reaction supplements

Current literature includes discussions on various PCR buffers and supplements, such as DMSO, PEG 6000, formamide, glycerol, spermidine and nonionic detergents, used to increase the reaction specificity or efficiency (15). Certain polymerases will only reach their optimum level of activity (16) in the presence of such supplements.

References

(1) Saiki, R.K., Scharf, S.J., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. & Arnheim, N. (1985). Enzymatic amplification of -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354.

(2) Persing, D.H.
'"/>

Source:

Page: All 1 2 3 4 5 6 7 8 9 10
Related biology technology :

1. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
2. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
3. Optimizing electroporation parameters
4. Optimizing Büchi® Rotary Evaporator Applications
5. Optimizing electroporation parameters
6. Optimizing Büchi® Rotary Evaporator Applications
7. Optimizing Bchi Rotary Evaporator Applications
8. Optimizing electroporation parameters
9. Optimizing electroporation parameters
10. Optimizing electroporation parameters
11. Optimizing electroporation parameters