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/ibcGetAttachment.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 (1 uL of 10 uM primer is often appropriate) || 0.2 - 0.3 μM ||
 * 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
 * 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: ＊ Felicia changes the 72 C step to 10 s. I have a Primestar Max protocol setup under my folder on the thermocycler computer.
 * 98 ℃ |||| 10 sec. ||
 * 55 ℃ |||| 5 sec. or 15 sec. || 30 - 35 cycles ||
 * 72 ℃ |||| 5 sec./kb ||

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.

-see also comparison of primestar max with dynazyme 7-3-13 -see also does it make a difference to do a hot start with primestar max 7-14-13 -see also template conditions appropriate for plasmid pool nested pcr with primestar max 7-31-13 --"F:\kurt\storage\CIM Research Folder\DR\2013\7-31-13\template conditions appropriate for plasmid pool nested pcr with primestar max 7-31-13.txt" -see also: template conditions appropriate for cDNA 7-31-13 "F:\kurt\storage\CIM Research Folder\DR\2013\7-31-13\template conditions appropriate for cDNA 7-31-13.txt"