containing mtff1 cdna lefebvre 1993 using the oligonucleotide primers mtff1s and mtff1a the mtff1s primer corresponds to the first 18 nucleotides of the sense strand of mtff1 from the first nucleotide behind the signal sequence the mtff1a primer is complementary to the last 26 nucleotides of the sense strand of mtff1 including the stop codon and introduces an extra spei restriction site pcr amplification was carried out using vent u2122 dna polymerase new england biolabs beverly usa which gives a pcr product carrying blunt ends the pcr mixture consisted of 2 units vent dna polymerase 10 u03bcl vent buffer thermopol 4 u03bcl dxtp 39 s 0 5 mm maximum 5 u03bcl 0 5 u03bcm of each primer 1 u03bcl 50 ng template dna and 74 u03bcl h 2 o six reactions were set up differing in their final concentration of mgso 4 adjusted to 0 1 2 3 4 and 5 mm respectively pcr amplification cycles were t 0 for 300 u2033 at 94 u00b0 c t 1 for 45 u2033 at 94 u00b0 c t 2 for 30 u2033 at 60 u00b0 c t 3 for 20 u2033 at 72 u00b0 c t 4 for 10 u2033 at 20 u00b0 c these cycles t 1 until t 3 were carried out 30 times pcr amplification with these primers rendered the gene for mature mtff1 lacking the signal sequence and including an additional spei restriction site after checking by gel electrophoresis the amplified fragment appeared as a band in the expected length range the 5 u2032 end of the mtff1 sequence contains two possible target sequences complementary to the forward.