Results of DNA Horses before Columbus research by geneticist Alessandro AchilliRuben LLumihucci
The entire horse mtDNA was amplified in 11 overlapping PCR fragments, using a set of oligonucleotides with matching annealing temperatures (Table S10). Oligonucleotides were checked (through GenBank BLAST) in order to avoid amplification of nuclear insertions of mitochondrial sequences (numts) (1). After PCR, the fragments were purified using the ExoSAP-IT® enzymatic system (Exonuclease I and Shrimp Alkaline Phosphatase, GE Healthcare) and standard dideoxysequencing was performed by using a set of 33 nested primers (Table S11) specifically designed for this protocol. An ABI 3730 sequencer with 96 capillaries was employed for separation of the sequencing ladders. Complete sequences were aligned, assembled, and compared using the program Sequencher 4.9 (Gene Codes). Traces were generally of excellent quality and there was extensive overlap between reads with most observed mutations determined by at least two independent sequencing reactions. At least two independent operators read each sequence and any potentially ambiguous base call was tested by additional and independent PCR and sequencing reactions.
Results of DNA Horses before Columbus research by geneticist Alessandro AchilliRuben LLumihucci
The entire horse mtDNA was amplified in 11 overlapping PCR fragments, using a set of oligonucleotides with matching annealing temperatures (Table S10). Oligonucleotides were checked (through GenBank BLAST) in order to avoid amplification of nuclear insertions of mitochondrial sequences (numts) (1). After PCR, the fragments were purified using the ExoSAP-IT® enzymatic system (Exonuclease I and Shrimp Alkaline Phosphatase, GE Healthcare) and standard dideoxysequencing was performed by using a set of 33 nested primers (Table S11) specifically designed for this protocol. An ABI 3730 sequencer with 96 capillaries was employed for separation of the sequencing ladders. Complete sequences were aligned, assembled, and compared using the program Sequencher 4.9 (Gene Codes). Traces were generally of excellent quality and there was extensive overlap between reads with most observed mutations determined by at least two independent sequencing reactions. At least two independent operators read each sequence and any potentially ambiguous base call was tested by additional and independent PCR and sequencing reactions.
Aardman Animations - The making of Pirates!Melanie Peck
Gavin Strange, founder of JamFactory and senior designer at Aardman Animations, gives a whistle stop behind the scenes tour of the making of the new film 'Pirates'.
Aardman Animations - The making of Pirates!Melanie Peck
Gavin Strange, founder of JamFactory and senior designer at Aardman Animations, gives a whistle stop behind the scenes tour of the making of the new film 'Pirates'.
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