Primestar Max DNA Polymerase

2015-01-13

azim58 - Primestar Max DNA Polymerase


PrimeStar Max
System from Takara/Clontech

Note that PrimeStar Max is the same thing as CloneAmp HiFi PCR

http://takara.co.kr/file/manual/pdf/R045A_e.v1108Da.pdf
or
http://www.clontech.com/takara/US/Products/PCR_Products/High_Fidelity_PCR/i
bcGetAttachment.jsp?cItemId=47286&fileId=6128473&sitex=10031:22372:
US
or
"F:\kurt\storage\CIM Research Folder\DR\2012\11-29-12\Primestar max
manual\PrimeSTAR Max DNA Polymerase Product Manual.pdf"

PrimeStar Max also seems to be good for vector linearization (see
Preparation of a Linearized Vector by PCR)


PrimeSTAR Max Premix last seen in Andrey's strategic reserve box -20 C
I also have some aliquots here: 7-2-13 -20 C box

General Composition of PCR Reaction Mixture

Final conc.

PrimeSTAR Max Premix (2X)
25 μl
1X

Primer 1
10 - 15 pmol (1 uL of 10 uM primer is often appropriate)
0.2 - 0.3 μM

Primer 2
10 - 15 pmol
(1 uL of 10 uM primer is often appropriate)
0.2 - 0.3 μM

Template
< 200 ng (1-10 ng is often appropriate)
＊

Sterilized distilled water
to final reaction volume of 50 μl


PCR Conditions
For reactions in which the quantity of template is 200 ng / 50 μl or
less:＊

98℃
10 sec.

55℃
5 sec. or 15 sec.
30 - 35 cycles

72℃
5 sec./kb


Note that before running large volumes of PrimeSTAR Max product on a gel, it is necessary to do a pcr purification. Otherwise the bands on the gel will look warped.



Felicia changes the 72 C step to 10 s.
I have a Primestar Max protocol setup under my folder on the thermocycler
computer.


===========================================================================

Note that there are special guidelines in the manual for using PrimeSTAR
max with cDNA
Here is the note about cDNA from the manual:

===========================================================================
For rapid amplification protocols (extension step of 5 to 10 sec./kb)
with cDNA as template, use a quantity of template that is to equal to or
less than the equivalent of 125 ng of total RNA / 50 µl reaction.
If larger quantites of cDNA template are desired, by setting a longer
extension time (up to 1 min./kb), it is possible to use up to the
equivalent of 750 ng total RNA / 50 µl reaction.

===========================================================================

One can start the PCR with a small amount of primer and later add more.
see PCR by starting with a small amount of primer and later adding more
primer and polymerase portal 4-11-13

With PrimeSTAR Max perhaps a reasonable procedure would be to add 1 uL of
2 uM each primer (0.04 uM final concentration each) and do 25 cycles.
Then add 0.5-1 uL 10 uM each primer (0.14 uM total concentration each
primer at this point), 5 uL H20, 5 uL PrimeSTAR Max Premix, and perform 5
more cycles, save an aliquot (10 uL), do 5 more cycles, save an aliquot,
and do this until there are no longer any more aliquots to save.


===========================================================================


7-14-13
with primestar max 7-31-13
conditions appropriate for plasmid pool nested pcr with primestar max
7-31-13.txt"
"F:\kurt\storage\CIM Research Folder\DR\2013\7-31-13\template conditions
appropriate for cDNA 7-31-13.txt"