Clontech PT3139-1 Gebruikershandleiding - Pagina 16

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CLONTECH Laboratories, Inc.

V. Troubleshooting Guide continued

Touchdown PCR

needed

Contamination

C. Products are smeared

Too many cycles

Denaturation temp. Try increasing the denaturation temperature in incre-

too low

Extension time

too long

Poor template

quality

Touchdown PCR

needed

Too much enzyme

[Mg

] is too high

Too much template Try a lower concentration of DNA template in the

Contamination

page

Protocol # PT3139-1

Version # PR76834

"Touchdown" PCR significantly improves the speci-

ficity of many PCR reactions in various applications

(Don et al. , 1991; Roux, 1995). Touchdown PCR

involves using an annealing/extension temperature

that is several degrees higher than the T

primers during the initial PCR cycles. The annealing/

extension temperature is then reduced to the primer

for the remaining PCR cycles. The change can be

performed either in a single step or in increments

over several cycles.

See Section D below.

Reduce the cycle number by 3–5 to see if non-

specific bands go away.

ments of 1°C.

Decrease the extension time in 1–2-min increments.

Check template integrity by electrophoresis on a de-

denaturing agarose gel. Repurify your template if

necessary.

See "Touchdown PCR needed" under previous

section.

Advantage-HF Polymerase Mix is 50X for most appli-

cations; however, a 1X final concentration of the

enzyme mix may be too high for some applications.

If smearing is observed, first try optimizing the cycle

parameters as described above, then try reducing

the enzyme concentration to 0.5–0.2X.

KlenTaq-1 DNA polymerase has a broader Mg

optimum than native Taq DNA polymerase (i.e., it

performs well over a wider range of [Mg

loss of efficiency.) Therefore, as long as you have

used the buffers supplied in the kit, it is unlikely that

smearing is due to problems with the Mg

concentration. Altering the concentration of Mg

result in lower fidelity.

PCR reaction.

See Section D below.

Technical Support TEL:415-424-8222 or 800-662-CLON

FAX: 415-424-1064 or 800-424-1350

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