---+ Primer Design http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=413997&INPUT_SEQUENCE=NC_012967.1&log$=seqview_list_primer Note: Following this link website automatically fills in certain fields with values specific to the strain. Going to the website independently (i.e., without following the link) requires manual input of this data. ---+++ Searching for Primers The product of the PCR process should include at least 100bp on either end of the sequence of interest. For example, if this sequence ranged from 1,000,000 to 1,001,000, then the region 999,900 to 1,001,100 should not be bonding to primers. To this end, enter ranges for the forward and reverse primer which do not cover this region. A descent range to begin with is 100bp. For example, if the region between 999,900 and 1,001,100 was to be copied, then a good initial search might be: Forward Primer: 999,800 to 999,900 Reverse Primer: 1,001,100 to 1,001,200 Larger ranges result in longer search times, but tend to find more primer combinations. It will often be necessary to extend the ranges far outside the ends of the sequence of interest in order to find compatible primers. Generally speaking, it is better to have the primers too far away than too close to the sequence of interest. However, it is best for the sequence of interest to be centered on the product. As long as the final product is less that 2500-3000bp, there is no major problem with wide sequences. Note that even the link from this website puts the product size limit to 1000bp. If a larger product is needed, this value must be manually changed. ---+++ Excluding Inappropriate Primers Once several primers have been found, analyze the candidates. If multiple primer combinations fit, as they often will, selecting the first one is quite acceptable. The first thing to check for is if the primer attaches to any sites outside of the intended target. Fortunately, the website states in bold when this is the case, and any primers which do bind outside of the intended region should be disregarded. Next, primers with multiple binding sites within the intended region should also be disregarded. Only primers which have only 1 location for the forward primer and 1 location for the reverse primer to bind should be taken into consideration. ---+++ Ordering Primers Follow the instructions at http://barricklab.org/twiki/bin/view/Lab/ProtocolsOrderingPrimers.
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Topic revision: r4 - 2011-06-03 - AurkoDasgupta