In the protocols I've found, important quantities are either omitted (volumes of DNA w/o concentration) or useless to me (0.5 flies of DNA). I'd like to know values people have found to work for: mean fragment size of digested DNA, concentration of DNA in ligation reaction (by mass or fragment #), and specificity of primers.
http://www.protocol-online.org/prot/Molecular_Biology/PCR/Inverse_PCR/
I am trying to identify where a randomly inserted cassette ended up - the sample will later be sequenced.
Ok I got you, well.. ok.. there are many ways to do this.. if you provide more information about the situation I can provide more detail... fragment size varies..depending on the situation.. anywhere from two bands to a smear...regarless.. you want to ligate with high volume reaction 50-200uL with a VERY LOW concentration of dna maybe .5ng/uL at final concentration in ligation rxn..
Primers.. there really isnt really anything special about this.. you want to get them as long as you can.. to try and reduce misprinting.. if you want more info.. you got to give more info
if in inverse pcr only reverse primer is amplifying the sequence at non target region..what is the solution of that?
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