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However if it’s still there after trying a gradient of temperatures, you may need to try a different pair of primers Don’t go changing out the primers just because you’ve got one appearing on your gel. There are other causes for the dreaded Primer Dimer band, and it can often be fixed by adjusting your annealing temperature. This will result in a thick band between ~30-100 bp mark on your agarose gel and little/no product of the desired size.
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Most plasmids will have standardised primers associated with them.Ĭertain primer pairs may be non-complementary because they associate more easily with each other than your target DNA strand/plasmid. Primer design is a challenging art and involves a bit of luck. Ideally use well established and understood primers when first learning PCR. I generally order these as single stranded DNA oligos from IDT. This will come packaged with a commercial polymerase.įragments of DNA, ~20-30 base pairs in length, which will be used to start off the polymerase chain reaction in each cycle. Provides a native environment that tricks DNA polymerase into working. Stolen from nature, this protein will be stitching the DNA together from the raw nucleotides, based upon the template you provide. ‘Rule of Thumb’ Annealing temperature = Tm - 5 You need primers that have a relatively similar Tm or you’ll get a lot of non specific product You want an annealing temperature somewhere around the Tm
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Tm = Temperature at which 50% of primers will be bound, and 50% unbound In theory, PCR products up to ~20 kb are possible, but these are extremely difficult to obtain in practice. Length of Amplicon = Distance between the 5’ ends of each primerĮxtension time = Number of Nucleotides to Amplify (bp) ÷ Speed of polymerase (bp/min)Īnything larger than ~5 kb is going to be difficult to amplify, even for an experienced user – success here requires using a fancy polymerase, *excellent* primers (well-designed, no dimers/hairpins), clean and high-quality template with high signal:noise ratio, fresh reagents, and excellent hands-on technique. I recommend you use Snapgene or Benchling dna Map of the Region you plan to replicate, complete with information about primer binding, and length of amplicon
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