Clontech PT3139-1 Gebruikershandleiding - Pagina 15

Blader online of download pdf Gebruikershandleiding voor {categorie_naam} Clontech PT3139-1. Clontech PT3139-1 20 pagina's. Advantage-hf pcr kit

Pagina afdrukken
V. Troubleshooting Guide continued
Too little enzyme
2+
[Mg
] is too low
[dNTPs] not optimal The Advantage-HF PCR Kit contains a carefully
Difficult target
B. Multiple products
Too many cycles
Annealing temp.
too low
Suboptimal primer
design
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Advantage-HF Polymerase Mix is 50X for most appli-
cations. Therefore, try to optimize the cycle param-
eters as described above before increasing the en-
zyme concentration. In rare cases, the yields can be
improved by increasing the concentration of the
enzyme mix. However, increasing the concentration
>2X is likely to lead to higher background levels.
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 use the
buffers included in the kit, it is unlikely that a lack of
product is due to problems with the Mg
However, if the concentration of EDTA in the cDNA
sample is more than 5 mM, this can reduce the effective
concentration of Mg to below a minimum level. Increas-
ing the concentration of Mg
fidelity.
balanced mixture of the 4 dNTPs. Replacement of
this mixture with a standard dNTP mix (200 µM each)
may improve the DNA yield, but may also result in
lower fidelity.
Some targets are inherently difficult to amplify. In
most cases, this is due to unusually high GC content
and/or secondary structure. The Advantage-GC Kits
are recommended in these situations.
Reducing the cycle number may eliminate nonspe-
cific bands.
Increase the annealing/extension temperature in
increments of 2–3°C.
Redesign your primer(s) after confirming the accu-
racy of the sequence information. If the original
primer(s) was less than 22 nt long, try using a longer
primer. If the original primer(s) had a GC content of
less than 45%, try to design a primer with a GC
content of 45–60%.
Technical Support
CLONTECH Laboratories, Inc.
2+
] with no
++
concentration.
2+
can result in lower
Protocol # PT3139-1
Version # PR76834
2+
page
15