PCR+Protocol

PCR Protocol

Designing PCR primers
 * primer3 can be used
 * It is good to have 1 or 2 Cs or Gs at the 3' end of the primer
 * usually a Tm (annealing temperature) around 55 C is desired
 * see Andrey's preferred method for calculating Tm in CIM

100 mM dNTPs located in freezer 62 -20 C

There are many different PCR Systems Advantage 2 PCR Protocol Colony PCR Protocol Primestar Max DNA Polymerase (this is a very fast polymerase) iProof polymerase (this is the most common polymerase used in our lab) Dynazyme (this is a fairly good polymerase that is also very cheap): Dynazyme Protocol Dynazyme Colony PCR Protocol Taq PCR Protocol Pfu (this is a very high fidelity polymerase) Pfu Ultra Polymerase Pfu Turbo Polymerase Herculase II Polymerase

Optimizing PCRs -one can try to adjust the template amount, annealing temperature (gradients are often performed), as well as cycle number

PCR by starting with a small amount of primer and later adding more primer and polymerase portal 4-11-13

Nested PCR

Touchdown PCR

Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences.

The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments of temperature decrease is chosen by the experimenter). -General conditions for touchdown pcr

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http://bitesizebio.com/articles/touchdown-pcr-a-primer-and-some-tips/

The suggested cycling program has two phases. The first phase of touchdown programming uses a Tm that is approximately 10C above the calculated Tm. The temperature is reduced by 1C every successive cycle until the calculated Tm range is reached. This is done for a total of 10-15 cycles. Phase 2 follows generic PCR amplification of up to 20-25 cycles using the final annealing temperature reached in the touchdown phase. The cycles and temperature drop during touchdown phase can be adjusted from 1-3 cycles per 1-3C drop in temperature if non-specific products are still observed or if the yield is low.

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-Hot Start PCR

Hot Start PCR is a modified form of Polymerase chain reaction (PCR) which avoids non-specific amplification of DNA by inactivating the taq polymerase at lower temperature. In conventional PCR, the Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice. In some instances, when all the reaction components are put together, nonspecific primer annealing can occur due to these low temperatures. This nonspecific annealed primer can then be extended by the Taq DNA polymerase, generating nonspecific products and lowering product yields.

Hot Start PCR can be performed by using specific antibodies which block the Taq-polymerase at annealing temperature.

Alternatively, the polymerase can be added post 1 min of denaturation.

http://www.researchgate.net/post/How_should_I_clean_up_this_PCR_smear

In Silico PCR

In Silico PCR can be used to predict bands that would be amplified from a PCR

UCSC In-Silico PCR http://genome.ucsc.edu/cgi-bin/hgPcr?db=hg18 This tool seems to work well for predicting P

-see Information about DNA Gels -see Optimization and troubleshooting PCR Kenneth Roux 2003 http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full